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COPYRIGHT DEPOSIT 



A TEXT-BOOK 



OF 



URINE ANALYSIS 



FOR 



STUDENTS AND PRACTITIONER 
OF MEDICINE. 



JOHN H. LONG, M.S., Sc.D., 

Professor of Chemistry and Director of the Chemical laboratories in the 
Schools of Medicine and Pharmacy of Northwestern University. 



WITH NUMEROUS ILLUSTRATIONS. 



EASTON, PA.: 
THE CHEMICAL PUBLISHING COMPANY, 
I900. 



TWO COPIES RECEIVED, 

Library of Cm*m«% 

MAY 8 -1900 

"•gltt.r of Cooyrtrtt* 
SECOND COPY, 






62642 

Copyright, 1900, by Edward Hart. 






48GS* 

o 



PREFACE 



In the following pages the subject of practical urine 
analysis is presented in a concise manner adapted to the 
requirements of the medical student in systematic class 
work, and also to the wants of the medical practitioner, 
who has occasion to make something more than the usual 
simple qualitative tests in urine examinations. A con- 
siderable number of quantitative methods are carefully £$ 
described, and in such a manner, it is believed, that any 
one with a slight previous training in the manipulation of 
chemical apparatus can follow them. On the other hand 
methods of doubtful applicability are consistently ex- 
cluded. 

This book contains much of the matter which appeared 
some years ago in the author's " Chemical Physiology and 
Urine Analysis." That work being out of print, the 
chapters on urine analysis have been rewritten and en- 
larged to appear, with additional chapters, in the present 
form. Two chapters are devoted to the microscopic ex- 
amination of sediments. 

While the book is essentially one of analysis, not of 
diagnosis, numerous references are made throughout the 
text to the clinical significance of what is found by the 
various tests, and in the appendix a section is devoted to 
a tabular statement of the relation of pathological condi- 



IV 



PREFACE. 



tions to the chemical composition of the urine. For many 
valuable suggestions in this direction the author is in- 
debted to his colleague, Dr. Frank S. Johnson. He must 
also acknowledge the assistance of Dr. Frank X. Walls in 
the preparation of illustrations, and of Mr. Frank Wright 
in the reading of proof. 

The Author. 

Chicago, March, /goo. 



TABLE OF CONTENTS 



Chapter I. 
Outline of Tests. Preliminary Tests I 

Chapter II. 

The Tests for Albumins • • ■. 16 

Chapter III. 
The Tests for Sugar, Acetone, Acetoacetic Acid, and Oxybutyric 

acid 48 

Chapter IV. 
The Coloring-matters in Urine. Biliary Acids 87 

Chapter V. 
Determination of Uric Acid. Hippuric Acid 105 

Chapter VI. 
Urea 119 

Chapter VII. 
The Determination of Phosphates, Chlorides, and Sulphates 145 

Chapter VIII. 
Ammonia, Xanthin and Allied Bodies, and Creatinin 167 

Chapter IX. 
The Sediment from Urine 175 

Chapter X. 
Unorganized Sediments and Calculi 201 

Appendix. 
Tables and Notes 223 



URINE ANALYSIS 



Chapter I 

OUTLINE OF TESTS. PRELIMINARY TESTS 

The importance of an accurate knowledge of the 
bodies excreted by the urine has long been recognized 
and elaborate investigations have been carried out to 
determine the nature and quantities of these substances, 
some of which appear normally in health, while others 
are found only during the progress of disease. 

Experiment shows that normally certain products 
occur in the urine in relatively large amounts, and 
give to it its prominent characteristics, while of other 
products the amounts present are so minute that their 
detection is a matter of no little difficulty. 

Certain grave disorders are accompanied by the 
appearance of certain substances in the urine, and 
where the chemical or microscopic tests for the latter 
are simple and unquestionably correct we have at hand 
a convenient aid to diagnosis. In many cases, how- 
ever, it is true that we are unable to trace the relation 
between small amounts of substances occasionally ap- 
pearing in urine and any specific disorder or condition 
of the body. The detection of such substances is nat- 
urally without value in diagnosis, at the present time. 



2 URINE ANALYSIS 

Yet it would be unwise to neglect the study of such 
traces because, as medical science progresses, new rela- 
tions are from time to time brought to light which 
give value to data which at one time may have been 
considered wholly unimportant. Complete handbooks 
on the urine give prominence to many topics which 
will not be touched upon in what follows because we 
are here concerned with phenomena, everywhere rec- 
ognized as important and the bearings of which, in 
the main at least, are understood. 

In the practical analysis of urine, such as is custom- 
ary for clinical purposes, comparatively few tests are 
required, and little apparatus is necessary beyond that 
already used for other examinations. Frequently a 
single test is sufficient to determine all the physician 
needs to know ; for instance, regarding the presence 
or absence of sugar or albumin. 

In the following pages those tests and processes 
will be described which have been shown by experi- 
ence to be amply sufficient for all practical require- 
ments. Some of these are qualitative, others quanti- 
tative, and may be tabulated as follows : 

i. Observation of color and odor. 

2. The reaction, whether acid or alkaline. 

3. The tests for albumin. 

4. The tests for sugar. 

5. The tests for the characteristic biliary acids and 
coloring-matters. 

6. The various tests for blood. 

7. Tests for other coloring-matters. 

8. The examination of the sediment. 



Cv 



PRELIMINARY TESTS 



. 


9- 


Determination oi 


4-> 








10. 




*-> 






<D 


n. 




*• 


12. 




ctf 






•iH 


13- 




c 






OS 

3 


14. 




O* 


L 15 - 





the amount of albumin, 



susrar. 



uric acid. 
urea. 

phosphates, 
chlorides. 



The above includes the usual and important tests. 
A few others will be given in the proper place ; for 
instance, tests for acetone and diacetic acid, which 
under circumstances may have importance. 

Normally, urine contains as its most important 
constituents urea, sodium chloride, certain phosphates 
and urates, and smaller amounts of other substances as 
hippuric acid, xanthin, creatinin, traces of phenols, etc. 

Several writers have given the results of complete 
urine analyses in tabular form. These results are not 
very concordant, as might be expected from the char- 
acter of urine itself. Two tables often quoted will be 
given here for comparison. 

According to Thudichum the average volume of 
urine passed per day is 1400 to 1600 cc, and the solid 
matter contained in the daily excretion he gives as 
follows, for a man weighing about 140 pounds. 



Urea - 

Uric acid 

Creatin and creatinin 

Hippuric acid 

Cryptophanic acid - 

Biliary acids 

Acetic acid 



Grams. 

30-40 

O.50 

0.75 

O.50 

O.65 

O.OI2 

O.288 



4 URINE ANALYSIS 

Grains. 

Formic acid - - - - - - 0.05 

Sulphuric acid, SO a - - - - 2.00 

Other sulphur combinations - - - 0.20 
Alkaline phosphates - - - - 3.66 

Earthy phosphates - - - 1.28 

Lime - - - - - - - 0.17 

Magnesia - - - - - - 0.19 

Potassium and sodium chlorides - - 1 1 . 50 

Ammonia - - - - - - 0.70 

Traces of other bodies are given by Thudichum, 
but in amounts too small for determination. 

Another table given by Parkes presents a better 
arrangement, and is here given ; the figures refer 
to the amounts excreted in twenty-four hours by a 
man weighing about 145 pounds. 





Grams. 


Urea ----- 


- 33-i8 


Uric acid ----- 


o.55 


Hippuric acid 


- 0.40 


Creatinin ----- 


0.91 


Organic acids and pigments 


- 10.00 


Sulphuric acid, S0 3 


2.01 


Phosphoric acid, P 2 5 


- 3-i6 


Calcium ----- 


0.26 


Magnesium - 


- 0.2I 


Potassium ----- 


2.50 


Sodium _ _ _ - 


- II.O9 


Chlorine ----- 


7-50 


Ammonia - 


" O.77 



It is evident that the character of the urine depends 
very largely on the diet, and this is shown in a clear 
manner by the figures in the following table, which 
were obtained by Bunge by the analysis of the urine 
of a healthy man, fed first on a meat diet, and later 



PRELIMINARY TESTS 



on one of wheat bread with salt and butter. Water 
was freely drunk in both tests. 





Meat diet. 


Bread diet 




cc. 


cc. 


mie in twenty-four hours. 


1672 


1920 




Grams. 


Grams. 


Urea - 


- 67.2 


20.6 


Creatinin 


2.163 


0.961 


Uric acid 


■ 1-398 


0-253 


Sulphuric acid, S0 3 


4.674 


1.265 


Phosphoric acid, P 2 5 


■ 3-437 


1.658 


Lime - 


0.328 


o- 39 


Magnesia 


■ 0.294 


0.139 


Potash - 


3-3o8 


i-3H 


Soda - 


3-991 


3.9 2 3 


Chlorine - 


3-8i7 


4.996 



These figures represent, of course, extreme cases, 
but their practical importance is readily recognized. 

Pathologically there may appear albumin, sugar, 
blood, pus, bile pigments and acids, and a number of 
other bodies insoluble, or of slight solubility, which 
usually appear as a sediment. 

We turn now to an explanation of the various pre- 
liminary tests employed. 

Specific Gravity 

The density or specific gravity of the urine secreted 
in twenty-four hours, varies in health between rather 
wide limits, probably between 1.005 an( ^ 1*030. 1.020 
may be taken as about the mean value at 15 C. 

The specific gravity depends primarily on the 
amounts of liquid and solid food taken, and on the 
loss of water from the skin by perspiration. When 



ITRINE ANALYSIS 



II 



this loss is great the specific gravity of the urine is 
correspondingly increased, other things being equal. 

In disease the density may be lowered below or in- 
creased above the normal value. 

For an absolutely exact determination of the density 
a the use of the pycnometer, Mohr- 

Westphal balance, or other ap- 
paratus is necessary, but for our 
purpose the urinometer, or den- 
sity bulb, is sufficiently accu- 
rate. This little instrument is 
shown in the adjoining figure. 
The urine to be tested is poured 
into a narrow jar, about one cm. 
wider than the bulb, and after 
the air bubbles have escaped 
the urinometer is immersed in 
it. When it comes to rest the 
degree at which it stands is 
read off below the siwface. Usu- 
ally the last two figures only of 
the density are marked on the 
stem, as 25, instead of 1.025, 
and these are often given as 
the density. 

As the density of urine de- 
creases about one degree for an 
increase in temperature of 3 to 
Fi g- r - 5 C. it is important that the test 

be made at a definite known temperature, as 15 or 
25 C. Urinometers have usually been graduated to 



PRELIMINARY TESTS 7 

give the correct reading at a temperature of 15.5 C. 
(6o° F.). But at the present time we have them for 
the temperature of 25 C. (77 F.), because this is with 
us a more common house temperature than the lower 
one. It is convenient to have the instrument indi- 
cate the correct specific gravity without the necessity 
of cooling. The specific gravity of urine varies ap- 
proximately as does that of water, with changes of 
temperature. A table in the appendix shows the rate 
for water, and by the use of this a correction can be 
made. 

By noting the amount of urine passed in twenty- 
four hours, and the density of the mixed liquid, a rough 
determination of the solid matters contained in it can 
be made. For this purpose it is simply necessary to 
multiply the last two figures of the density by 2.33 
(known as the coefficient of Haeser\ which gives the 
approximate number of grams in a liter. By propor- 
tion the amount for the day can be calculated from 
this. 

For example, 1,400 cc. of urine was passed, and its 
density was found to be 1.024, 
Then, 24 X 2.33 = 55.92, 
and, 1,000 : 1,400 : : 55.92 : x = 78.288. 

This calculation is frequently of service. 

As indicated above a variation in the specific gravity 
of normal urine may be due to several causes, the 
most important of which are changes in the volume 
of water drunk or the weight of nitrogenous food and 
salt digested. The amount of urine excreted daily 
may be taken as 1,500 cc. in the mean. Assuming 



8 URINE ANALYSIS 

that 15 grams daily is the salt consumption and that 
it is all excreted with the urine we have through this 
factor alone a specific gravity of about 1.007. Assu- 
ming further that 150 grams of nitrogenous food (con- 
sidered as pure albumin) are consumed daily and that 
four-fifths of this amount is daily excreted as urea, the 
weight of the latter in the urine would be 43 grains. 
This alone would produce a density of nearly 1.008 
and give a percentage composition of 2.84. Combined 
with the other substances in urine the relative effect 
of the addition of urea would be o-reater. All of the 
solids of the urine have a specific gravity greater than 
that of water and their presence therefore adds to the 
specific gravity of the excretion, but changes in the 
density, due to changes in the amounts of uric acid, 
phosphates, sulphates, etc., passed, are of less impor- 
tance because of the relatively small quantities of these 
substances normally present. 

If, with the food consumed normal, the water taken 
is small in amount, or if a large amount is lost from 
the skin as perspiration then the density of the excre- 
ted urine must be correspondingly higher. A large 
volume of water consumed or little evaporation from 
the skin will give a urine of lower density. It is 
plain, therefore, that great variations in the specific 
gravity of the urine may occur and from perfectly nor- 
mal causes. 

In disease even greater variations may occur, one of 
the most characteristic and important being that due 
to the presence of sugar in diabetes mellitus. Here 
the density may reach 1.040 or higher, while the vol- 



PRELIMINARY TESTS 9 

time of urine is above 1,500 or 2,000 cc. in the twen- 
ty-four hours. A high specific gravity with large vol- 
ume is always suspicious and suggests presence of dex- 
trose, although occasionally it may be due to presence 
of large doses of soluble salts taken into the system as 
remedial agents. A low specific gravity with small 
volume of urine must also call for investigation, as this 
points to the absence of, or marked decrease in, the nor- 
mal constituents from some cause. A lower density 
is observed in diseases where the elimination of urea 
is slower because of hindered tissue changes, in con- 
ditions of malnutrition in general, and in any disease 
involving the structure of the liver itself. In acute 
yellow atrophy of the liver, for instance, urea is much 
diminished, and the specific gravity low. 

The diminution in excreted chlorides, with normal 
consumption, in certain diseases is also a factor in 
causing low specific gravity. This may follow when 
the salt consumed is eliminated temporarily in various 
exudations or effusions rather than by the normal 
channel. 

Some of these indications will receive attention 
later during the discussion of the tests for the common 
normal and abnormal urine constituents. 

Reaction 

In health the reaction of the mixed urine passed 
through twenty-four hours is always acid. 

This normal acid reaction is supposed to be due to 
the presence of acid phosphates and to small amounts 
of uric acid and to other free organic acids. The 



IO URINE ANALYSIS 

reaction can be observed by the aid of sensitive litmus 
paper, but the absolute amount of free acid is very 
small. 

Occasionally urine is passed which gives the so- 
called amphoteric reaction with litmus ; that is, it 
turns blue paper red and red paper blue. It has not 
been found possible to connect this phenomenon with 
certainty with any definite pathological condition ; it 
has, therefore, no special clinical significance at the 
present time. Some hours after a hearty meal an 
alkaline reaction is frequently observed, giving place 
soon to the usual acid condition. This alkaline reac- 
tion may be due to the presence of small amounts of 
trisodium phosphate formed during active digestion. 
The administration of alkaline carbonates, or of 
certain organic salts, as malates, acetates, tartrates, or 
citrates, which yield carbonates by final decomposition, 
may also occasion an alkaline condition. It should 
be observed that the character of the food consumed 
has much to do with the quality of the urine as 
regards acidity or alkalinity. In the consumption of 
products rich in proteids the oxidation of the sulphur 
gives rise to sulphuric acid which converts the alkali 
phosphate of the blood into acid phosphate before excre- 
tion. With a diet low in proteids the acidity is greatly 
decreased. Sometime after it is voided urine always 
becomes strongly alkaline in reaction, but this change 
may be delayed for days or weeks even. It is brought 
about by the decomposition of urea, which is usually 
a result of bacterial action. In this decomposition 
ammonium carbonate is formed, the odor of which 



PRELIMINARY TESTS I I 

often becomes very strong. Anything which prevents 
or impedes the bacterial activity tends to maintain the 
ordinary acid or neutral reaction. Salicylic acid, 
thymol, chloroform, volatile oils, and other antifer- 
ments behave in this manner, and are frequently added 
to specimens of urine to preserve them for investigation. 
For the preservation of ioo cc. of urine one-fourth 
of a gram of salicylic acid is enough. 

Sometimes the decomposition of the urea takes 
place in the bladder, the voided urine having then a 
very marked alkaline reaction and strong odor, usually. 
Such a change may be brought about by the progress 
of disease, or may be induced by the introduction of 
a dirty catheter into the bladder. This carries the organ- 
ism capable of splitting up the urea, and the condition 
once established may be maintained for a long time. 

Ammonium carbonate results from the reaction. 
This may be distinguished from the fixed alkalies 
(hydroxide or carbonate of sodium or potassium) by a 
very simple test. A piece of sensitive red litmus 
paper immersed in alkaline urine becomes blue. On 
drying the paper the color due to the non-volatile 
alkalies persists, while that of ammonium carbonate 
disappears. The test has some practical value as it is 
necessary to distinguish between the alkalinity of urea 
fermentation and that of an excess of fixed alkali 
occasionally present. For these tests only fresh sensi- 
tive paper can be safely used. The conversion of urea 
into ammonium carbonate is represented by this equa- 
tion : 

CON„H 4 + 2H 2 = (NH 4 ) 2 C0 3 . 



12 URINE ANALYSIS 

In highly colored urines it is not always easy to 
observe the reaction with litmus paper. In this case 
a method often used in the examination of blood to 
determine its alkalinity may be applied. This con- 
sists in immersing small disks of plaster of Paris in 
neutral litmus solution and then drying them. A few 
drops of urine are placed on a disk and allowed to 
remain some minutes. The urine is then washed off 
leaving a bluish or reddish spot indicating the 
reaction. 

An accurate determination of the acidity of urine 
can not be readily made, but approximately a value 
may be obtained by adding a few drops of phenol- 
phthalein to ioo cc. of urine, and then running in 
tenth-normal sodium hydroxide solution until a per- 
manent pink color is secured. At this stage the acid 
phosphates of the type H 2 NaPO are converted into 
the alkali phosphates of the type HNa 2 P0 , and for 
each cubic centimeter of alkali used 12 milligrams of 
H 2 NaPO may be calculated as present. But on 
account of the color of the urine itself the process is 
at best somewhat uncertain. 

To approximately determine the alkalinity add to 
100 cc. of urine a few drops of phenolphthalein, and 
then tenth-normal sulphuric or hydrochloric acid until 
the color disappears. If the alkalinity is due to 
sodium carbonate, for each cc. of the acid used, calcu- 
late 5.3 milligrams of Na 2 C0 as present. If due to 
Na PO each cc. of alkali used corresponds to 8.2 
milligrams. All such tests should be made in a 
beaker with a wide bottom placed over white paper so 



PRELIMINARY TESTS 1 3 

as to disclose a change of color as clearly as possible. 
For the preparation of the standard acid and alkali 
solutions used in these tests see the appendix. 

Odor 

The odor of urine is not easily described, as in 
health it is sui generis and characteristic. Normal 
urine contains traces of complex aromatic bodies, the 
exact nature of which cannot in all cases be given. 
These substances are more abundant after a vegetable 
than after an animal diet, and are especially note- 
worthy in the urine of persons whose food contains 
such vegetables as cabbage, radishes, parsnips, aspara- 
gus, or the spices. It is well known that certain sub- 
stances given as remedies give rise to distinct odors in 
the urine. The administration of turpentine imparts 
to the urine an odor of violets. 

As the odor so largely depends on the nature of the 
food it may be much modified even in health, and in 
disease may be characteristically changed. The am- 
moniacal odor of urea decomposition in the bladder 
has been referred to, and the peculiar sweetish odor of 
diabetic urine has long been noticed. 

But it must be remembered that many strong odors 
may be developed in the urine soon after passage by 
the action of ferments other than the micrococcus 
urea which yields ammonium carbonate. In some 
cases these give rise to what may be called a putre- 
factive odor. 

Color 

The color of urine is described as straw-yellow. 



14 URINE ANALYSIS 

Many causes, however, may produce a change in this 
shade, leaving the urine still normal. As can be 
readily seen the color is closely dependent on concen- 
tration and must, therefore, vary with the amount of 
liquid taken into the stomach. 

Certain foods from the vegetable kingdom possess 
characteristic coloring-matters which pass, more or 
less changed, into the urine. As long as the latter is 
acid the presence of these may not be noticed, but 
w r ith a change of reaction a change of color may 
follow, usually to reddish. 

Santonin imparts a yellowish color to urine, red- 
dened by alkalies. In pathological conditions the 
color of urine is often characteristic and of great im- 
portance in diagnosis. The presence of blood, for 
instance, is indicated by a more or less sharp shade of 
red, bile by a peculiar greenish brown, especially 
noticeable in froth produced on shaking. The urine 
of diabetes mellitus is generally very pale, while the 
urine of fevers is usually highly colored not only from 
the diminution of water, but also from the presence of 
abnormal coloring-matters. Different shades are pro- 
duced by the presence of altered blood and bile con- 
stituents, which will be referred to later. The real 
color of the urine is often obscured by loss of trans- 
parency due to precipitation. Normal urine is gener- 
ally perfectly transparent when passed, but sometimes 
cloudy from presence of a suspended precipitate of 
mucus or phosphates. On becoming alkaline a pre- 
cipitation of earthy phosphates usually follows. A 
precipitate of urates, without change of reaction, often 



PRELIMINARY TESTS 



15 



takes place by simply lowering the temperature of the 
urine. This precipitate, however, disappears on the 
application of a slight heat to the urine, and leaves 
the latter clear for examination of color. 

Clinically, the color indications of greatest import- 
ance are those due to the presence of derivatives from 
the blood or bile. These may be unaltered elements 
of the blood or bile or decomposition products of their 
essential coloring-matters. 

In a following section on the tests for abnormal 
coloring-matters in urine this question will be again 
taken up. 



Chapter II 

THE TESTS FOR ALBUMINS 

Albuminous bodies do not occur in normal urine 
except, perhaps, in mere traces. Numerous investi- 
gations have been published on this subject, and while 
some of the recent ones would seem to show the prob- 
ability of a physiological albuminuria, others, seem- 
ingly as thorough, lead to quite the opposite conclu- 
sion. 

Temporarily, it is true, albumin may be found in 
the urine of healthy individuals, as after the consump- 
tion of large quantities of egg albumin, or after 
the action of some cause producing a sudden altera- 
tion of the blood pressure, but the amounts found in 
such cases are too small, and their occurrence too rare, 
to permit them to be classed as anything but acci- 
dental. It is certain that the presence of any appre- 
ciable amount of albumin in the urine and the per- 
sistence of the same must be looked upon as a patho- 
logical phenomenon and one of the greatest impor- 
tance to the physician. 

Albumins may appear in urine from several sources, 
most frequently, probably, because of some structural 
change in the tubules of the kidneys which permits a 
filtration from the blood. But this is not always the 
case as they may appear from sources in no way de- 
pendent on renal disorder, from lesions of the ureters, 



TESTS FOR ALBUMINS 1 7 

bladder, or urethra, for instance, in which case blood 
or pus may be present. Ordinary serum albumin is 
the usual, but not the only proteid body which may 
appear in urine. Half a dozen or more modifications 
have been described as occurring under different cir- 
cumstances, but the evidence for some of these appears 
to be of doubtful character. Certain forms are not 
readily detected or identified. In what follows, tests 
will be given for those proteid bodies which can be 
detected with certainty and whose presence has some 
definite clinical importance. 

i. Serum Albumin 

The presence of serum albumin in the urine is a 
characteristic of what is ordinarily termed albuminuria. 
As intimated above albuminous bodies may appear in 
the urine from different sources. The presence of 
serum albumin suggests (a) a functional or structural 
disorder of some part of the essential tissue of the kid- 
ney, in which case we have renal albuminuria or true 
albuminuria, or (b) a lesion of some part of the uri- 
nary tract below the kidney, in which case we have 
what is called false or accidental albuminuria. 

Renal albuminuria is the condition appearing in 
Bright's disease or acute parenchymatous nephritis and 
in other pathological conditions in which a change of 
the diffusion membrane is involved. It is also fre- 
quently induced by derangements in the circulation 
due to heart diseases, high fevers, etc., which in turn 
may react and give rise to a derangement of the kid- 
ney itself. That is to say, the causes producing cer- 



1 8 URINE ANALYSIS 

tain febrile conditions may extend to the structure of 
the renal filtering apparatus and so alter its condition 
that the passage of albumin is no longer hindered but 
becomes continuous. 

Under all such circumstances the albumin passing 
through the kidney is generally accompanied with 
something which suggests its origin. There may be 
here an excessive amount of the epithelial lining of 
the tubules, or plugs of coagulated albumin, mucus, 
or of the wax-like, partially degenerated albumin 
known as lardacein, all in the form of "casts" of the 
uriniferous tubules. These may be readily seen and 
recognized by the microscope. 

As intimated above, false or accidental albuminuria 
can originate from several causes and in general is a 
condition of far less clinical importance than the other. 
It is usually possible to determine by a few examina- 
tions the real nature of the disorder by aid of the facts 
just mentioned. 

Because of the very great importance of the subject 
to the physician, much attention has been given to 
the question of albumin tests, and the number of re- 
actions proposed for its detection reach, possibly, the 
hundreds. Many of these are of such extreme delicacy 
and so easy of execution that to make a choice of a 
few is by no means a simple matter. 

The best of them depend on the fact that the solu- 
ble serum albumin, which finds its way into the urine, 
can be coagulated and made visible as white flocculi, 
or as a white cloud when present in small quantity. 
Of the various methods of producing this coagulation, 



TESTS FOR ALBUMINS 1 9 

only those will be mentioned which are most charac- 
teristic, and practically the most useful. 

Qualitative Coagulation Tests 

Coagulation by Heat. — When a sample of urine is 
boiled, a precipitate usually forms. This in most cases 
consists of earthy phosphates, and is often sufficient to 
conceal a precipitation of albumin possibly present. 
If now to the boiled sample about one-tenth its vol- 
ume of strong nitric acid be added, the precipitated 
phosphates will disappear, while the albumin will re- 
main coagulated. It is necessary to add as much nitric 
acid as is here indicated, because a small amount may 
sometimes dissolve coagulated albumin, forming solu- 
ble acid-albumin. This acid-albumin is broken up on 
the addition of more acid. 

Even when boiling does not throw down a precipi- 
tate, the addition of nitric acid cannot be omitted, as 
under certain circumstances the heating may produce 
a soluble combination between alkalies present and 
albumin, which is stable. Nitric acid in sufficient 
quantity will break up this combination and bring 
about coagulation. 

Under most circumstances this heat test, as outlined, 
is sufficient, and the possibility of making a mistake is 
very small. It is shown in works on chemical physi- 
ology that small amounts of albumin combine readily 
with weak acids and alkalies, forming soluble and 
stable combinations known as acid-albicmin and alkali- 
albumin. 

If the urine has a neutral or alkaline reaction 



20 URINE ANALYSIS 

to begin with a small amount of alkali-albumin 
would escape detection by heating alone. On addition 
of just the proper amount of acetic acid to neutralize 
the alkali, the application of heat will cause a coagu- 
lation, but a slight excess of this acid might convert 
the alkali-albumin into acid-albumin, equally hard to 
precipitate. Traces of nitric acid, and in a marked 
degree hydrochloric acid, behave in the same manner, 
but the addition of larger amounts of nitric acid is 
free from this objection because in proper amount this 
acid is able to decompose both acid- and alkali-albumin. 

When taken for examination, urine is frequently 
cloudy from the presence of precipitated urates or 
earthy phosphates. Heat is sufficient to dissipate the 
cloud if due to the urates, but the phosphate cloud is 
rendered heavier. It is always a good plan to care- 
fully filter the urine, if in the least degree turbid, before 
undertaking the test. 

With old samples of urine which have undergone 
the urea fermentation and have become alkaline, the 
test by heat and subsequent addition of acid is not 
always satisfactory or convenient. In such cases it is 
best to proceed at once to a method which disposes of 
the excess of alkali at the start, and in such a manner 
as to cause no confusion. 

Coagulation by Nitric Acid. — As indicated above ni- 
tric acid can coagulate albumin, and this test is fre- 
quently employed without previous boiling. When 
applied to fresh urine the test may be made in this 
manner. 

Several cc. of the strong acid are warmed in a test- 



TESTS FOR ALBUMINS 21 

tube, and over this is carefully poured an equal vol- 
ume of urine, so as to overlie without mixing. If 
albumin is present a white ring appears at the surface 
between the two liquids. When the urine contains 
an excess of coloring-matter the ring is variously 
tinted. 

If urine is poured over cold acid, a precipitate may 
appear which is not albumin. This can happen when 
the urine is highly charged with urea, in which 
case crystalline nitrate of urea will separate out, or 
where urates are abundantly present, in which case 
the ring will consist of very fine crystals of uric acid, 
or acid urates. Both of these precipitates are dissi- 
pated by heat, and if the nitric acid is previously 
warmed, they cannot appear. It is better to make the 
test as just suggested than to use cold acid, and then 
try to warm a ring formed, as this would cause an 
admixture of the liquids sufficient to obscure a slight 
amount of albumin. 

It is sometimes recommended to pour the urine in 
a test-tube, and by means of a pipette, or dropping- 
tube, allow the acid to flow under it. This is an ex- 
cellent method of performing the test, but the acid 
should be slightly warm as before. If only a trace of 
albumin is present the ring will not appear immedi- 
ately, but only after standing. It is well, therefore, 
in doubtful cases, to set the tube aside for twelve hours 
and then observe it. If a ring is now found it should 
be very gently and carefully warmed to determine its 
behavior toward heat, because on standing in the cold 
a ring of urates might appear. 



22 URINE ANALYSIS 

When this test is applied to old, cloudy, or alkaline 
urine it should be preceded by this preliminary prep- 
aration : 

Boil the urine with half its volume of 10 per cent, 
potassium hydroxide solution and filter. This will 
usually give a bright, clear liquid, but if not, add two 
drops of the "magnesia mixture" employed in quali- 
tative analysis and described in the appendix, boil and 
filter again. The filtrate is now suitable for testing. 

The action of the reagents is this : The strong al- 
kali forms a bulky precipitate of the earthy phosphates 
present which usually settles and leaves the supernatant 
liquid clear. The amount of alkali taken is sufficient 
to prevent the coagulation and precipitation of the 
albumin on boiling, while it serves also to expel am- 
monia which may be present. If the first filtrate is 
not perfectly clear the addition of the magnesia mix- 
ture accomplishes this by making a new precipitate 
of phosphates in traces which now leaves it bright. 

With the clear filtrate the tests by addition of nitric 
acid may now be carried out. It must be remembered, 
however, that as the urine is now strongly alkaline, a 
relatively large volume of the strong nitric acid must 
be employed. 

The text-books abound in minute descriptions con- 
cerning the best methods of conducting this compara- 
tively simple test. The few sources of error which 
may mislead will now be pointed out. It is, of course, 
understood that these appear only in the search for 
small amounts of albumin; that is, for amounts less 
than one-tenth of i per cent, by weight. For greater 



TESTS FOR ALBUMINS 23 

quantities the reactions, even when not conducted 
with extreme care, are usually sharp. 

When urine is poured over nitric acid or when the 
acid is introduced under the urine* a layer of some kind 
always appears at the junction of the two liquids. 
The problem is to decide what this is. The peculiar 
appearance of a relatively large amount of coagulated 
albumin is so characteristic that any one who has ever 
seen it will recognize it again. But a faint cloud or 
haziness is, at the start, somewhat confusing. A 
colored layer or ring, which is very common, must 
not be mistaken for a precipitate ' or cloud. The 
normal urine coloring-matters may produce a highly 
colored ring, and the bands with biliary colors are 
even deeper. But these color bands or zones are 
transparent which can be determined by holding the 
test-tube in the proper light. 

Urine very highly charged with urea may give a 
crystalline precipitate of urea nitrate. This is a very 
unusual reaction, and the precipitate may be very 
easily recognized through the form and size of the 
crystals, which are large flat plates readily seen by the 
naked eye or by a common magnifying lens. If a 
urine suspected to contain such an excess of urea be 
diluted with an equal volume of water before testing 
the crystals will not appear. Besides, they do not 
appear when the liquids are warm. The finely gran- 
ular precipitate of acid urates or hydrated uric acid 
appears only in a cold liquid, therefore cannot be 
present to mislead if the test is conducted as directed. 

If the special tests indicate the presence of unusually 



24 URINE ANALYSIS 

large quantities of urates the urine may be diluted with 
an equal volume of water before adding the nitric acid. 
It occasionally happens that a yellowish white cloud 
or band appears in this test which is not due to 
albumin or uric acid. Such a cloud may be caused 
by the presence in the urine of bodies taken into the 
system as remedies and which are excreted in but 
slightly changed form. Derivatives of turpentine 
and certain resinous bodies are specially liable to 
behave in this manner. After the use of copaiba 
balsam, nitric acid throws out from the urine insoluble 
resin acids which are not dissipated by heat. The 
precipitate formed by such acids dissolves easily in 
alcohol and can thus be readily distinguished from 
albumin which is not soluble. It must be remem- 
bered that while pure albumin precipitated by nitric 
acid is white, that thrown down from urine may be 
more or less colored from the presence of normal or 
abnormal coloring-matters. 

Attention must be called to a method of conducting 
the nitric acid test which is frequently employed, but 
which for small quantities of albumin is very untrust- 
worthy. This method consists in mixing about equal 
volumes of strong acid and urine, and boiling. This 
is open to the grave objection that by it the albumin 
sought may be decomposed and so lost from view. 
Nitric acid is a very strong oxidizing agent, and 
albumin a substance easily decomposed. Traces may 
therefore be lost even by a very short boiling, as may 
be readily determined by the student by a few experi- 
ments with weak albumin solutions. 



TESTS FOR ALBUMINS 25 

Tanret's Mercuric-Potassium Iodide Test. — A solu- 
tion of this compound precipitates albumin from 
acidified urine and is on the whole an extremely deli- 
cate reagent. Among the general albumin tests em- 
ployed by chemists for the recognition of proteids it is 
always shown that many of these bodies are thrown 
out from their solutions in the form of complex basic 
compounds by addition of salts of certain heavy metals. 
Soluble salts of mercury, lead, and copper give charac- 
teristic reactions. 

Tanret prepared a well-known and popular test solu- 
tion in the following manner : 

Dissolve 33.12 grams of pure potassium iodide in 
about 200 cc. of distilled water. Add 13.54 grams of 
powdered mercuric chloride and warm until, with suffi- 
cient stirring, the red precipitate of mercuric iodide dis- 
appears, leaving a clear slightly yellowish solution. Di- 
lute this with distilled water to about 800 cc, and add 
100 cc. of pure, strong acetic acid. Allow to stand over 
night if not absolutely clear, and decant from any 
small precipitate which may have settled out. Dilute 
then to one liter with distilled water. This solution 
contains the two salts in the proportion of 4KI to 
HgCl,. 

The test with the reagent so prepared is carried out 
as follows : 

Filter the urine to make it perfectly clear, and add 
enough acetic acid to give it a good acid reaction. To 
about 10 or 15 cc. in a test-tube add a very little of 
the reagent, a drop at a time, from a pipette or drop- 
ping-tube. In all not more than five drops should be 



26 URINE ANALYSIS 

added, as this is sufficient to give a strong precipitate 
if albumin is present. The precipitate is flocculent, 
and appears as a white cloud or streak, as the first drop 
of the heavy mercuric solution settles and mixes with 
the urine. As each following drop mingles with the 
urine the hazy cloud grows to a precipitate in case the 
urine contains more than a mere trace of albumin. 

The delicacy of the reaction is remarkable. It is 
said that by it one part- of albumin in one hundred 
thousand parts of urine may be detected. This, how- 
ever, is probably excessive. One part in twenty-five 
thousand in a series of tests is nearer the average re- 
sult. It has been claimed that where the solution is 
to be kept a long time it is best prepared without the 
addition of the acetic acid, as this is liable to produce 
slight decomposition in time. It is likely that the 
danger of this has been overestimated. 

In any event, unless the urine is fresh and slightly 
acid the addition to it of acetic acid should not be 
neglected. The use of the acid is said by some writers 
to be unnecessary, but it has the advantage of disclo- 
sing the presence of any quantity of mucin which might 
interfere with the test. If the acid throws out a cloud 
of mucin it should be filtered off and then the reag-ent 
added. 

While this is an exceedingly valuable test certain pre- 
cautions must be observed in its use. The mercuric 
solution is similar to one used as a test for alkaloids, 
and in fact precipitates many of these bodies. Qui- 
nine and other alkaloids given as remedies and excreted 
by the urine would therefore be shown by the test. 



TESTS FOR ALBUMINS 27 

Alcohol dissolves these precipitates, however, but is 
without solvent action on that formed by albumin. 
Uric acid and urates give precipitates with the reagent 
if present in large amount. These precipitates can be 
avoided by diluting the urine before testing, or if 
formed can be dissipated by slight heat. 

Mistaking mucin for albumin can be avoided as 
shown above. Small amounts of peptones are pre- 
cipitated by the reagent, but the coagulum disappears 
by application of heat. 

The Ferrocyanide Test. — A reagent of great delicacy 
is potassium ferrocyanide in presence of acetic acid. 
It shows not only serum albumin, but globulin and 
perhaps other proteids. It does not give a reaction 
with peptones. 

The test is applied in this manner. The urine must 
be made as clear and bright as possible by filtration 
and then strongly acidulated with acetic acid. If a 
precipitate or cloudiness from mucin appears now, filter 
again and to the filtrate add four or five drops of a 
fresh, clear solution of the ferrocyanide. With even 
traces of albumin this gives a flocculent yellowish 
white precipitate.- 

One of the advantages claimed for this test is that 
it gives no reaction with the vegetable alkaloids and 
therefore can be used as a check upon some of the 
others, the mercuric-potassium iodide test for instance. 
The precipitate formed, although flocculent, is very 
fine and can be observed therefore only in clear solu- 
tion. 



28 URINE ANALYSIS 

A modified form of the test is the following: Mix 
five drops of the ferrocyanide solution with 5 cc. of 30 
per cent, acetic acid. Pour this carefully over an equal 
volume of clear urine in a test-tube and allow to stand 
a short time. A white zone at the junction of the 
liquids show T s the albumin. 

The Picric Acid Test. — Picric acid solutions, pure, 
or combined with citric, acetic, or other acids, have 
long been used as reagents for the detection of traces 
of albumin in urine. In its simplest form the test 
liquid employed is a saturated aqueous solution of pure 
picric acid. It gives a very characteristic yellowish 
flocculent precipitate with even traces of albumin. 
Another solution frequently employed contains in one 
liter 10 grains of picric acid and 20 grams of citric 
acid. It must be made clear by filtration, if necessary, 
and is applied to clear urine in small quantity by 
means of a pipette, so as to show a cloudiness as the 
liquids mingle. The reagent is added gradually to 
the urine and in all not more than one-half the vol- 
ume of the latter for a qualitative test. 

The real practical value of this test is, in some quar- 
ters, still in dispute. It is certainly very delicate, but 
as it gives precipitates with peptones, alkaloids, urates, 
mucin, creatinin, and perhaps other bodies, the first 
result observed is subject to revision. The precipi- 
tates formed with these substances and picric acid are 
dissipated by heat, but there is risk of getting the 
temperature too high, in which case other pecipitates 
are liable to be formed, especially with the plain solu- 



TESTS FOR ALBUMINS 29 

tion without the citric acid. A urine which yields a 
precipitate of earthy phosphates by warming will give 
it at the same temperature in the presence of picric 
acid. It seems to be true, however, that with citric 
acid added the interference from phosphates is elimi- 
nated, and there remains only mucin as a disturbing 
element. The danger here is not great, and it is likely 
that in all cases it can be avoided by adding the citric 
acid first, filtering if necessary, and then adding the 
picric acid. 

Trichloracetic Acid Test. — A saturated aqueous solu- 
tion of this acid is a very delicate reagent for albumin. 
The test is made by pouring the solution on the urine 
in a test-tube. In presence of albumin a white cloud 
or zone appears at the junction of the two liquids. 
Other proteids besides serum albumin are coagulated 
by this reagent, but it appears to be without action on 
peptones or mucin, and on this account has come into 
favor among medical men. If much uric acid or an 
alkaloid is present in the urine, it is best to warm the 
latter before applying the reagent. 

Other Tests. — A number of other tests are in use 
which show very minute traces of albumin, but they 
seem to possess no advantages over those enumerated 
above. One of these depends on the precipitation of 
albumin by phenol and acetic acid; in another picric 
and hydrochloric acids are used; in a third a strong 
solution of common salt and hydrochloric acid; and so 
on, but practically no one will find it necessary to go 
beyond the six tests given. Indeed, two are by most 



30 URINE ANALYSIS 

authorities generally thought sufficient; viz., the heat 
test and the nitric acid test. What cannot be shown 
by these reactions is so minute that for practical pur- 
poses it can be neglected generally. 

The Amount of Albumin 

It is not alone sufficient that we are able to detect 
the presence of albumin in urine ; we often need to 
know 7 its amount to determine the practical value of a 
line of treatment pursued from day to da}'. To be of 
the greatest possible service, a method must be so easy 
of execution that approximately correct results may 
be obtained by it by the use of simple apparatus and 
in a short time. Several methods are known by which 
the amount of albumin in urine can be found. One 
of these, and the best, may be called the gravimetric 
method, as by it the albumin is precipitated, collected, 
and weighed. In another, the albumin is precipitated 
and its volume measured, while in a third process the 
amount of albumin is estimated from the degree of 
turbidity caused by its precipitation in the urine. 

Only the first and second methods will be described. 
The first is employed in exact investigations, and the 
second in clinical estimations. 

The Gravimetric Method. — If the qualitative test has 
shown only a small amount of albumin 100 cc. of the 
urine should be measured out into a beaker for pre- 
cipitation. If the qualitative test has given a strong 
indication 50 or 25 cc. should be taken and diluted to 
100. Enough dilute acetic acid is added to the urine 
to give it a faint acid reaction, after which it is brought 



TESTS FOR ALBUMINS 3 1 

up to a temperature of 80 ° or 90 ° C. on the water- 
bath, being stirred, meanwhile, frequently. From 
time to time the beaker is held up against the light, 
so that the operator may determine whether the coag- 
ulation is complete or not. A satisfactory coagulation 
is shown by the precipitation of the albumin in large 
flakes, leaving the surrounding liquid nearly clear. If 
this is not the case a little more acid should be added, 
but very carefully, and in all but three or four drops, 
unless the urine was strongly alkaline to begin with. 
When the reaction seems to be complete on the 
water-bath the beaker is placed on gauze and the con- 
tents brought to boiling. The precipitate is then al- 
lowed to settle. Meanwhile, a small filter of well- 
washed filter-paper is dried and weighed in a weigh- 
ing tube. It is plaited and put in a funnel and then 
the albumin precipitate is collected on it. The pre- 
cipitate is washed with hot distilled water until it gives 
no chlorine reaction to the wash-water, then with ab- 
solute alcohol, and finally with ether. The funnel 
with contents is placed in an air-oven and dried at 
120 C. The filter is transferred to the weighing 
tube, and when cold is weighed. The increase in 
weight gives the albumin. Instead of collecting on 
paper, a much better plan is to collect onaGooch fun- 
nel of asbestos, when it can be had. This simplifies 
the test, besides adding to its accuracy. This method 
consumes a good deal of time, but gives results which 
are near the truth when it is properly conducted. The 
best results are obtained when the weight of the pre- 
cipitate does not exceed 0.3 gram. 



32 



URINE ANALYSIS 



Volume Methods. — One of the simplest of these is 
the one proposed by Esbach. In this a special tube 
is used, called the Esbach albuminometer, 
and a special solution or reagent made by 
dissolving 10 grams of pure picric acid 
and 20 grams of pure citric acid in a liter 
ill I of distilled water. The solution must be 
filtered if it is not perfectly clear, and is 
the same as the one used for the qualita- 
tive test. The principle involved in the 
employment of the method is this : The 
precipitate of albumin and picric acid 
settles in coherent manner and in a com- 
pact volume proportional to its weight, 
provided certain definite amounts of the 
reagent and urine are taken. The albu- 
minometer, or measuring tube used, re- 
sembles a test-tube of heavy glass about 
six inches long, and is graduated, empiric- 
ally, to show how much urine and reagent 
to take and the amount of albumin ob- 
tained expressed in grams per liter, or 
tenths of 1 per cent. The annexed cut 
shows the tube and its markings. 

The test is carried out in this manner. 
Urine is poured in, to the mark, and 
then the reagent, described above, to its 
proper level. The tube is closed with 
the thumb and tipped backward and 
forward eight or ten times until the liquids are 
thoroughly mixed. It is then closed with a rubber 




Fisr. 2. 



TESTS FOR ALBUMINS 33 

stopper and allowed to stand in a perpendicular 
position twenty-four hours. This will give the pre- 
cipitate time to settle thoroughly after which the 
amount can be read off on the scale. The results are 
accurate enough for clinical purposes and by practice 
can be made to agree moderately well with those 
found by the gravimetric method. 

But to obtain this close agreement a number of pre- 
cautions must be observed. The volume of the pre- 
cipitate is in a marked degree variable with the tem- 
perature, and with the time given it for subsidence. 
The empirical graduation is based on the supposition 
that the test will be made at a temperature of 15 to 
20 C, and that the reading will be made at the end of 
twenty-four hours. If the reading; is delayed to two 
or three days the volume of the precipitate will be 
found much smaller. At the present time small cen- 
trifugal machines are rapidly coming into use to set- 
tle urine sediments. Some of these are operated by 
the Edison electric light current, and give the rota- 
ting tubes a high velocity. Where these machines are 
employed to settle the picric-acid-albumin precipitate, 
the volume of the latter may be rendered abnormally 
small and the reading, therefore, prove erroneous. 

The volume of the precipitate will depend here, not 
only on time and temperature, but also on velocity of 
rotation, and the effect of this factor must be deter- 
mined for each instrument before it can be accurately 
used. 

In any case in applying this test the urine should 
not be highly concentrated. The best results are ob- 
3 



34 URINE ANALYSIS 

tained with urine of low specific gravity and with the 
albumin not over o. 3 of 1 per cent. If a test shows an 
amount greatly in excess of this the urine should be 
diluted with a known proportion of water and tested 
again. On long standing in the cold, a yellowish red 
precipitate of uric acid sometimes settles out. This 
need not mislead the analyst, as its color and 
general appearance are quite distinct from those of 
albumin. 

The precipitates of albumin from urine by whatever 
means obtained are bulky and lead to the impression 
that the amount present is much larger than is actu- 
ally the case. It was at one time customary to speak 
of 25, 30, and 50 per cent, of albumin, these numbers 
representing the apparent volume of the precipitate 
in the test-tube. When these light precipitates are 
collected, properly dried, and weighed, a very different 
volume is obtained. A urine with 1 per cent, of albu- 
min contains an unusually large amount and in any 
case seldom more than 10 or 15 grams of albumin 
occur in the day's urine. Between 1 gram and 5 grams 
are more common amounts, even in cases of acute 
albuminuria. 

It will not be necessary here to discuss any of the 
other processes proposed for the rapid determination 
of albumin in urine. While some of them are certainly 
much more accurate than the Esbach method as just 
outlined, they are all too complicated for quick clinical 
manipulation, and in this respect are no better than 
the long gravimetric process. 



TESTS FOR ALBUMINS 35 

2. Serum Globulin 

This is an albuminous body resembling the serum 
albumin in many respects and often, perhaps gener- 
ally, associated with it. 

The globulins, as a class, are distinguished by their 
insolubility in pure water and very concentrated salt 
solutions, but they dissolve readily in weak salt solu- 
tions. Besides serum globulin there are recognized 
here crystalling of the crystalline lens, vitellin, found 
in the yolk of the egg, fibrinogen, of the blood which 
gives rise to fibrin on coagulation, myosin, found in 
the muscle substance after death, and finally globin, 
which is a proteid produced by the decomposition of 
hemoglobin of the blood. The discussion of these 
bodies belongs in the field of physiological chemistry. 

At the present time globulin in the urine has the 
same clinical significance as serum albumin. Although 
similar to each other in most points there are several 
characteristic differences which are taken advantage 
of as qualitative tests. The general relations of al- 
bumin and globulin are pointed out in text-books of 
chemical physiology where reactions are given for 
each. But not all of the tests for globulin which we 
find given in the books are suitable for use in the 
examination of urine, however, as we are here limited 
by the presence of other substances. Most of the re- 
actions given above for albumin apply equally well to 
globulin, and it is only within recent years that 
attempts have been made to detect one in the presence 
of the other. Among the methods applicable in the 
examination of urine the following may be given as 
most characteristic. 



36 URINE ANALYSIS 

Qualitative Tests 

Dilution Test. — Globulin is insoluble in water, but 
soluble in dilute salt solutions; — hence its solubility 
in urine. If the latter is diluted until the specific 
gravity is 1.002 or 1.003 tne globulin may separate 
out. At any rate the addition of a few drops of dilute 
acetic acid will produce the desired result. A current 
of carbon dioxide passed into the diluted liquid for 
several hours accomplishes the same end. 

The test may be modified in this manner. Filter 
the urine if it is not perfectly clear, and then pour it, 
drop by drop, into a tall, narrow beaker of distilled 
water. If globulin is present it is thrown out as a 
white cloud which shows as the drops pass down 
through and mix with the lighter, clear water. The 
globulin may afterward be confirmed by adding a 
small amount of salt solution which will cause the 
precipitate to disappear. 

Sulphate Test. — Globulin may also be detected by 
reason of its insolubility in strong salt solutions. To 
this end treat the urine with enough ammonia water 
to give an alkaline reaction. This precipitates phos- 
phates and sometimes other salts, which after a time 
are filtered off, leaving a clear liquid. To this add an 
equal volume of saturated solution of ammonium sul- 
phate which, in presence of globulin, produces a white 
flocculent precipitate. 

Magnesium sulphate is frequently used for the same 
purpose, and under some circumstances may possibly 
be preferable. 



TESTS FOR ALBUMINS 37 

In this test there is some danger of confounding 
albumose with globulin, as the former is also precipi- 
tated by ammonium sulphate. But the danger of 
confusion is small if the conditions given are adhered 
to ; i. e., mix equal volumes of the clear filtered urine 
and saturated ammonium sulphate solution. For the 
precipitation of albumose higher concentration is 
necessary, as will be further explained below. 

Where magnesium sulphate is used it is necessary 
to employ it in very considerable excess. The test is 
best made by adding the perfectly pure salt, in pow- 
dered form, to the urine until the latter becomes sat- 
urated. Under these conditions globulin, if present, 
separates. If the sulphate is not pure it may yield a 
turbid liquid on dissolving, in which the test is 
uncertain. 

The figures given in many of the text-books, as to 
the amount of serum globulin found in the urine, have 
in several instances been obtained by methods which 
are quite erroneous, and are therefore valueless. It 
appears from the most trustworthy investigations that 
in all cases of albuminuria globulin is present with 
the albumin, and in amount varying from 8 to 60 
per cent, of the whole proteid content. 

3. Albumose or Hemialbumose 

We have here a representative of an important class 
of proteid compounds which are derived from the 
albumins proper. It is well known that in the diges- 
tion of native albumins the albumoses appear as one 
of the stages, and are found, therefore, among the prod- 



38 URINE ANALYSIS 

nets of peptic and pancreatic action in the body. It 
is likely that normally albumoses are always converted 
into peptones before leaving the alimentary tract. 

The special significance of the bodies called albu- 
moses in the urine is by no means clear. While cer- 
tainly a pathological appearance it has not yet been 
found possible to definitely connect it with any one 
disease. Its presence has been reported in the urine 
in several cases of osteomalacia, but it appears by no 
means to be a constant accompaniment. Observers 
have called attention to its occurrence during the 
progress of several other diseases, but without being 
able to point out any definite relation. 

Qualitative Tests 

The recognition of albumose is not a matter of diffi- 
culty, as it is distinguished from the other proteid 
compounds sometimes found in the urine by several 
well-marked characteristics. It is not coagulated by 
heat or by the addition of acetic or warm nitric acid, 
and is very soluble in hot water. It is much less sol- 
uble in cold water, but the presence of small amounts 
of salts seems to increase its solubility here in marked 
degree. 

In presence of albumin or globulin it can be found 
by the following process unless it is in very small 
amount. The urine is saturated with pure sodium 
chloride, which will precipitate albumose if present, 
and then enough acetic acid is added to give a very 
strong acid reaction. The mixture is boiled and 
filtered hot. This treatment throws out both albumin 



TESTS FOR ALBUMINS 39 

and globulin, and redissolves a precipitate of albumose 
which may have formed. The latter would therefore 
be found in the clear nitrate and sometimes in amount 
sufficient to precipitate as this cools. The nitrate 
should therefore be allowed to remain at rest until 
quite cool. If much albumose is present it will ap- 
pear as a white cloud. Sometimes, however, it will 
be necessary to concentrate the nitrate before looking 
for this reaction, and this is done by evaporating slowly 
on the water-bath to half the volume. On now cool- 
ing, salt will quickly settle out while the albumose 
precipitates later in flocculent form. 

Another test is this : Separate the albumin and 
globulin by boiling with a small amount of acetic acid 
without the salt. Filter while warm and concentrate 
the filtrate to a volume of one-third. Allow to cool 
thoroughly and add a large excess of saturated solu- 
tion of ammonium sulphate. This gives a white floc- 
culent precipitate of albumose, if present. The pre- 
cipitate can be collected on a filter and washed with 
the saturated ammonium sulphate solution and then 
dissolved in a little distilled water, poured on the filter. 
This filtrate gives tests with picric acid, potassium fer- 
rocyanide and acetic acid, and other albumin reagents. 

If the original urine shows no reactions for albumin 
or globulin the albumose tests can be applied directly 
after concentration. The method by precipitation by 
means of picric acid gives good results. The biuret 
test is also delicate if the urine is clear and of light 
color. 

The amount of albumose in a urine cannot be found 



4-0 URINE ANALYSIS 

by any simple method suitable for clinical application. 
Several processes have been described, but they in- 
volve a determination of nitrogen by the Kjeldahl or 
other method, and can therefore be carried out only 
in well-equipped chemical laboratories. 

4. Peptones 

As has been explained, peptones are proteid com- 
pounds formed from native albumins, globulins, fibrin, 
etc., in the digestive process. In this respect they are 
related closely to the albumoses, both differing from 
the other proteids in important respects ; but pep- 
tones have further characteristic properties by which 
they are differentiated in turn from the albumoses. 

Peptones may occur in the urine as a result of va- 
rious abnormal conditions of the body ; but their ap- 
pearance does not depend, like that of albumin, on 
changes in the circulation or on pathological condi- 
tions of the kidney. Their clinical significance, there- 
fore, is very different from that of albumin or globulin. 

In cases of phosphorus poisoning the urine has fre- 
quently been found to contain peptones, but their 
presence can usually be connected with the disintegra- 
tion of pus somewhere in the body. The peptone 
substances are, therefore, frequently, or perhaps gen- 
erally, found in the urine in cases of purulent menin- 
gitis, purulent pleurisy, in the termination of pneu- 
monia by resolution, and in general under circum- 
stances in which products of suppuration can find 
their way into the systemic circulation to be eliminated 
afterward bv the kidnevs. 



TESTS FOR ALBUMINS 4 1 

Peptones have been reported in erysipelas, pulmo- 
nary tuberculosis, acute articular rheumatism, carci- 
noma of the gastro-intestinal canal, catarrhal jaundice, 
and in numerous other disorders. The condition in 
which the urine contains peptones as a result of the 
breaking up of purulent products is spoken of as 
pyogenic peptonuria, in contradistinction to that in 
which there is no indication of the existence of a sup- 
purative process. It is claimed that in certain can- 
cerous conditions of the stomach and intestine, the 
peptones of digestion may find their way into the cir- 
culation. 

Normally, peptones do not exist in the blood in more 
than traces, and during absorption from the healthy 
surfaces of the alimentary tract a change into albumin 
seems to take place. It is held by some writers that 
if taken up from an unhealthy surface (ulcerated) this 
conversion may not take place and peptone unchanged 
enters the circulation to disappear finally by way of 
the kidneys and through other channels. 

It has been shown also that peptones are a normal 
constituent of the urine of women in the puerperal 
state and their occurrence has been pointed out under 
still other conditions not connected with a suppurative 
process. However, in the great majority of cases in 
which their existence is shown the connection with 
the latter is clear, and the detection of these substances 
becomes important as an aid to diagnosis. 

In regard to the amount of peptone which may be 
found in the urine we have no very full data. About 
5 grams daily appears to be the maximum reported in 



42 URINE ANALYSIS 

cases of croupous pneumonia, but ordinarily the quan- 
tity remains far below this. 

Many reactions characteristic of peptones are given 
in the books, but not all of these may be applied to 
the urine. It should be further said that some of the 
proteid products described under the name peptone 
have probably no connection with that found in urine, 
and such bodies are not necessarily included in the 
tests given below. Among the characteristics of pep- 
tones in general the following may be recalled : i. 
They are extremely soluble in water and from such a 
solution they are not precipitated by boiling or by 
addition of acid. 2. They are insoluble in alcohol 
and may be precipitated by an excess from aqueous 
solution. This precipitate dissolves readily on the 
addition of plenty of water and is not a permanent 
coagulum. 3. Peptones are precipitated by acid so- 
lutions of phosphomolybdic acid, phosphotungstic acid, 
and by solutions of several heavy metallic salts. 4. 
The biuret test is given by peptones. In this test the 
liquid to be examined is made strongly alkaline with 
a solution of potassium hydroxide and then a few drops 
of copper sulphate solution are added. In presence of 
peptone the light rose-red or violet color is produced. 
Other forms of albumin behave in the same manner. 

Below a few T tests which may be applied to urine 
are given. 

Biuret Test. — The direct treatment of the urine 
with strong alkali and the copper sulphate solution is 
seldom sufficient, it being usually necessary to subject 



TESTS FOR ALBUMINS 43 

it to a preliminary treatment to remove substances 
which interfere with the reaction. A large amount of 
peptone substance unmixed with albumin or globulin 
may give a very characteristic color, but in a highly 
colored urine this may be unsatisfactory, and it is 
therefore safest to apply first a purifying process. 
The nature of the preliminary treatment will depend 
on the presence or absence of albumin or globulin. 
First, supposing these bodies absent we may proceed 
as in the following : 

Hofmeister's Tests. — In order to prove the presence 
of peptone in urine at least half a liter must be taken 
and treated with neutral lead acetate in amount 
sufficient to produce a heavy flocculent precipitate, 
which is separated by filtration. An excess of the 
lead must not be used, but the solution is added care- 
fully, a few drops at a time, giving the liquid mean- 
while opportunity to settle so that a fresh precipitate 
can be seen after the addition of more of the reagent. 
By using proper care the right point can be found 
when addition of the acetate must cease. 

Supposing now that we have a clear filtrate and 
that no albumin is present, we next add to the nitrate 
a little hydrochloric acid, and then a solution of 
phosphotungstic acid in hydrochloric acid as long as 
a precipitate forms. If peptone is present it is con- 
tained in this precipitate which must be separated 
immediately by filtration, and washed on the filter 
with dilute sulphuric acid (5 per cent.) until this passes 
through without color. The moist precipitate is then 



44 URINE ANALYSIS 

transferred to a small dish and mixed with a slight 
excess of barium carbonate or with enough crystalline 
barium hydroxide to give a slight alkaline reaction 
after thorough stirring. A little water is added and 
the whole is heated on the water-bath about ten 
minutes and filtered. The filtrate is then tested for 
peptone by the biuret test, or by the picric acid solu- 
tion, as used in other proteid tests. 

If albumin is present in the original urine it cannot 
be completely removed by lead acetate as just explained. 
The reaction with acetic acid and potassium ferrocya- 
nide will usually show it in the filtrate from the lead 
and it must be removed as follows, this being accom- 
plished by precipitating it with ferric oxide. Add to 
the 500 cc. of filtrate a small amount of sodium acetate 
solution and then some ferric chloride, after which 
the urine is made neutral with sodium hydroxide and 
boiled. The iron should be completely precipitated, 
carrying with it the albumin. The solution is filtered, 
allowed to cool, and tested for albumin. If free from 
this it is ready for the peptone test, beginning with 
the addition of hydrochloric acid. If albumin is still 
present, add a little more iron or alkali, as experiment 
will decide, and boil again. 

The phosphotungstic acid solution is made up by 
various formulas, but as a reagent for urine the fol- 
lowing method is recommended : A solution of pure 
sodium tungstate is made of about twenty per cent. 
strength. To this is added either glacial phosphoric 
acid or sirupy phosphoric acid and the mixture boiled, 
the acid being added in sufficient amount to give a 



TESTS FOR ALBUMINS 45 

strong acid reaction. With glacial phosphoric acid 
the proportion should be about one part to four of the 
tungstate. The boiled mixture is allowed to cool 
thoroughly, and to it is added about, one-fifth its 
volume of strong hydrochloric acid. Allow the 
mixture to stand and pour off the clear liquid from 
any precipitate which may settle out. The complex 
phosphotungstic acid is one of the few reagents which 
completely precipitate peptones and other albuminous 
bodies. In this case it is applied after the other 
albumins are thrown out by other means and serves to 
take . the peptone away from coloring and equally 
objectionable substances. 

Negative Tests. — Peptones are not precipitated by 
heat or by the addition of hydrochloric, sulphuric, ni- 
tric, or acetic acid. The reaction with potassium fer- 
rocyanide and acetic acid, characteristic for the other 
albumins, is not given by the peptones. 

They differ from the albumoses in not being thrown 
down by excess of ammonium sulphate, from which 
it follows that the test below can sometimes be applied 
for the detection of peptones in presence of albumin 
or albumose. 

Precipitate 50 to 100 cc. of urine, acidulated with a 
few drops of acetic acid, by boiling. Filter, concen- 
trate the filtrate to a volume of 5 cc, allow to cool and 
add 50 cc. of cold saturated solution of ammonium 
sulphate and then some of the pure crystals so that 
the whole liquid is completely saturated. Then filter 
and to the filtrate apply the biuret test with a large 



46 URINE ANALYSIS 

excess of alkali and a trace only of copper sulphate, or 
apply the phosphotungstic acid test. 

If the latter test is used the nitrate from the albu- 
mose should be diluted with two volumes of distilled 
water. The solution of phosphotungstic acid gives a 
precipitate with normal urine, and also with the un- 
diluted ammonium sulphate filtrate. 

But reduced with water as directed no precipitate of 
the ordinary urinary constituents appears. A slight 
opalescence only may result, while in presence of even 
traces of peptones there is a marked precipitate. The 
reaction between phosphotungstic acid and peptone 
solutions is one of extraordinary delicacy, so that traces 
show even after the above treatment. 

In making the biuret test in a solution containing 
a large amount of ammonium sulphate some color is 
always obtained. In absence of peptone it is the char- 
acteristic blue of the copper sulphate and ammonia reac- 
tion, but in presence of peptone the shade is reddish 

violet. 

5. Mucin 

In small amount mucin is probably present in all 
normal urines, in the case of women coming especially 
from the vagina. In moderate amount it has, there- 
fore, no pathological significance. If coming from the 
urinary tract in more than minute traces it usually 
indicates an irritated condition or catarrh of the pas- 
sages and then has clinical interest. 

Urine containing mucin in lar^e amount is turbid 
when passed ; with a smaller amount it may be clear 
at first but on standing deposits a cloud which settles 



TESTS FOR ALBUMINS 47 

nearly to the bottom of the vessel and there floats in 
loose form, instead of compact as with other sediments. 
This clond does not clear up by the addition of acetic 
acid or dilute nitric acid. 

In clear urine containing mucin a flocculent, hazy 
precipitate is formed by the addition of acid. The 
test is best made by pouring some acetic acid into a 
test-tube and then carefully an equal volume of urine 
so as to mix the two liquids as little as possible. A 
mucin cloud appears in the urine layer above the zone 
of contact of the liquids. An albumin cloud makes 
its appearance lower, or at the zone. In testing for 
mucin in presence of albumin the main portion of the 
latter should be precipitated first by boiling and filter- 
ing. The mucin test can be applied to the cold filtrate 
by addition of acetic acid. 

Mucin as well as albumin is precipitated from urine 
by the addition of an excess of strong alcohol (three 
volumes to one). After some hours the precipitate 
may be collected on a filter and washed with alcohol. 
It is then washed with warm water which dissolves 
the mucin. This may be recognized in the aqueous 
solution by the addition of acetic acid. 

Small amounts of mucin are so frequently mistaken 
for traces of albumin that attention must be paid to 
the methods of distinguishing between them. From 
what has been said it will be understood that the cloud 
which appears as a diffused haze in testing for albumin 
by an excess of acetic acid or a very small trace of nitric 
acid may be due to mucin and not to albumin as fre- 
quently assumed by mistake. A proper excess of nitric 
acid redissolves mucin, but not albumin in the cold. 



Chapter III 

THE TESTS FOR SUGAR, ACETONE, ACETOACETIC 
ACID, AND OXYBUTYRIC ACID 

On the question of the occurrence of sugar in the 
urine a vast amount has been written. At one time, 
indeed until within quite recent years, it was gener- 
ally assumed that normal urine contains no sugar or 
carbohydrate of any kind. But present methods of 
research seem to throw doubt on the truth of this 
view. It is not possible to separate small traces of 
sugar from a complex liquid like the urine so that the 
body separated may be recognized by its sensible prop- 
erties. On the contrary we must depend on the re- 
sults of certain reactions given by sugar solutions and 
in many instances by other organic bodies, and it is 
on the proper interpretation of these reactions that the 
authorities differ. Some of these reactions for traces 
will be explained below. In this place it suffices to 
say that the leading physiological chemists of the 
present time are nearly unanimous in holding that 
traces of the sugar known as dextrose exist normally 
in urine, in other words, that there may be such a con- 
dition as pJiysiological glycosuria as distinguished from 
the well-known pathological condition characterized 
by the presence of relatively large amounts of sugar 
in the urine and named diabetes mellitus. 



THE TESTS FOR SUGAR, ACETONE, ETC. 49 

The amount of sugar believed to be normally pres- 
ent is very small and cannot be recognized by the first 
three or four tests given below. An amount of sugar 
in the urine sufficient to have clinical importance is 
readily recognized by many tests. 

The characteristics of urine in true diabetes are 
these : It has a specific gravity higher than normal, 
usually between 1.030 and 1.040, and this with a 
greatly increased quantity. A high specific gravity 
with small volume, it has been shown, need have no 
special clinical importance as such a condition can re- 
sult from many causes outside of disease. Diabetic urine 
is usually light in color and prone to speedy decom- 
position by fermentation. The amount of sugar which 
can be present in advanced stages of diabetes mellittis 
may be very large. It is said that as much as 1,000 
grams of dextrose has been passed with the urine in 
one day in extreme cases ; but the amount usually 
coming under the observation of the practitioner is far 
below this, 10 to 100 grams being much more com- 
mon amounts. In typical diabetes the percentage 
amount of the normal urine constituents is usually 
greatly diminished because of the great dilution with 
water, but the actual amount excreted in twenty-four 
hours may be increased. 

It is well known that sugar may temporarily occur 
in the urine from a variety of causes. It has been 
found after the absorption of several poisons, and in 
cases of carbon monoxide poisoning ; also in the 
course of certain diseases. 

The amounts present in these circumstances are 
4 



50 URINE ANALYSIS 

usually small, aud disappear with other symptoms of 
the disorder. The continued presence of considerable 
quantities of sugar is characteristic of only one disease; 
i. e., the diabetes mellitus. This fact should be borne 
in mind in the practical examination of urine and 
tests should be repeated from time to time, unless the 
other clinical evidence is sufficient to immediately 
confirm the indication of the chemical test. 

Qualitative Tests 

The tests for sugar in urine depend on several dis- 
tinct general reactions. The most common of these 
reactions is that due to the oxygen absorbing power 
of alkaline dextrose solutions. The absorption of 
oxygen may give rise to a solution of characteristic 
color and odor as in the first one of the tests given 
below, or to certain precipitates formed by the ab- 
straction of oxygen from metallic combinations in the 
test solutions employed. 

This oxygen absorbing or reducing power of sugars 
in general is due to their peculiar chemical structure, 
inasmuch as the}' must be regarded as the aldehyde or 
ketone derivatives of polyhydric alcohols. Common 
dextrose is probably an aldehyde, and therefore active 
in its reducing power. The sugar tests which are 
most commonly employed in urine analysis will be 
explained at some length because of their great im- 
portance. 

Moored Test. — This depends on the reaction between 
grape sugar and strong alkali solutions. When a so- 
lution of sugar or diabetic urine is mixed, without 



THE TESTS FOR SUGAR, ACETONE, ETC. 5 1 

heating, with a solution of sodium or potassium hy- 
droxide, no change is at first apparent unless the 
amount of sugar present is large or the alkali very 
strong. But on application of heat, even with weak 
sugar solutions, a yellow color soon appears which 
grows darker, becoming yellowish brown, brown, and 
finally almost black, while an odor of caramel is quite 
apparent. The strong alkali-sugar solution absorbs 
atmospheric oxygen, giving rise to a number of prod- 
ucts among which lactic acid, formic acid, pyrocate- 
chin and others have been recognized. The brown 
color is due to other unknown decomposition products. 

This is a good reaction for all but traces of sugar, 
as the intense dark brown color and strong odor are 
not given by other substances liable to be present in 
urine. 

But traces of sugar cannot be recognized by this 
test with certainty, as the color of normal urine even 
is darkened to some extent by the action of alkalies. 

Urine containing much mucin becomes perceptibly 
darker when heated with sodium, potassium, or cal- 
cium hydroxide solutions. 

The Trommer Test. — This is one of the oldest and 
best known of the tests for the recognition of sugar in 
urine, and has been referred to before. It is performed 
by adding to the urine an equal volume of 10 per cent, 
solution of sodium or potassium hydroxide and then a 
very few drops (three or four to begin with) of dilute 
solution of copper sulphate. 

Solutions of alkali and copper sulphate alone give 



52 URINE ANALYSIS 

a blue precipitate of copper hydroxide, but in presence 
of sugar and certain other bodies a deep blue solution, 
and not a precipitate, is formed. Therefore, if the 
urine tested contains sugar the first indication is a 
more or less blue solution, stable for some time in the 
cold. On standing, however, the liquid turns green- 
ish, and finally deposits a yellow precipitate. This 
change takes place immediately on application of heat, 
the greenish-colored precipitate turning yellow, and 
finally red by boiling. Copper suboxide precipitates, 
and this is the second and characteristic stage of the 
Trommer reaction. 

Several substances can give the first stage, but dex- 
trose is the only body liable to be present in the urine 
which can give a good indication in the second. 

The test must, however, be used with certain pre- 
cautions. Albumin, if present, must be coagulated 
and filtered off. The amount of copper sulphate used 
must be small, because if only a trace of sugar is pres- 
ent and much copper is used the latter will give a blue 
precipitate which does not redissolve, and which turns 
black on boiling, thus obscuring a sugar reaction 
which may be given at the same time. 

In adding the copper sulphate it is best to pour into 
the test-tube containing the urine and alkali, first 
about three drops of a five per cent, solution. If this 
appears to give a yellow color on boiling, which does 
not turn black, more should be added, and this con- 
tinued until a yellow or red precipitate is formed. A 
black precipitate on boiling shows that too much cop- 
per has been added, and that probably sugar is absent. 



THE TESTS FOR SUGAR, ACETONE, ETC. 53 

The active body in producing the reaction is copper 
hydroxide, but this must be in solution to act as a 
good oxidizing agent with sugar ; and the test, there- 
fore, becomes uncertain or unsatisfactory if so much 
copper is added that the hydroxide formed cannot be 
dissolved by the sugar which may be present. In 
doubtful cases it becomes necessary to make several 
trials before the right proportion between urine, alkali, 
and copper solution is found. In the solution, on 
completion of the reaction, several oxidation products 
of sugar are found, among which are formic acid, 
oxalic acid, tartronic acid, etc. But the complete re- 
action is obscure. In order to avoid the indicated un- 
certainty of the Trommer test when used for small 
amounts of sugar the next one was proposed. 

The Fehling Solution Test. — Fehling suggested the 
use of a solution containing along with the copper 
sulphate and alkali a tartrate to dissolve the copper 
hydroxide formed by the first two. Many substances 
besides sugars, referred to in the last paragraph, have 
the power of dissolving copper hydroxide with a deep 
blue color. Among these may be mentioned tartaric 
acid and the tartrates, glycerol, mannitol and others 
of less value. 

A solution prepared by mixing certain quantities of 
alkali, copper sulphate, and either one of these bodies, 
with water in definite proportions remains perfectly 
clear when boiled. But if a trace of dextrose (or sev- 
eral other sugars) is present the usual yellow precipi- 
tate forms. 

In the preparation of the original Fehling solution 



54 URINE ANALYSIS 

it was assumed that, in the presence of alkali, the re- 
action takes place between exactly five molecules of 
copper sulphate and one of dextrose, and on this as- 
sumption the solution was made of such a strength 
that i cc. would oxidize five milligrams of dextrose. 
We have, therefore, the relation : 

5(CuS0 4 . 5 H 2 0) : C 6 H 12 6 

1248 : 180 : : 34.66 : 5. 

Each cubic centimeter of the prepared Fehling solu- 
tion must contain 34.66 milligrams of pure crystal- 
lized copper sulphate and just how to best combine 
this with the necessary alkali and tartrate is shown in 
the appendix. 

The great advantage which this solution has over 
the Trommer test is found in the fact that it may 
always be used safely in excess. With only a trace 
of sugar there is no danger that the copper will pre- 
cipitate as black hydrated oxide. In performing the 
test a few cubic centimeters of the Fehling solution 
(4 or 5) are poured into a test-tube, diluted with an 
equal volume of water, and boiled. The solution must 
remain clear. Then the urine is poured in, at first 
about half a cubic centimeter, and the mixture boiled. 
If sugar is present in amount above one-tenth of 1 per 
cent, it should show with the volume of urine taken. 
For smaller amounts of sugar more urine must be 
added, and the mixture boiled again. 

Wnen normal urine is heated with Fehling solution, 
a greenish flocculent precipitate usually makes its ap- 
pearance. This has no significance as it is due to the 



THE TESTS FOR SUGAR, ACETONE, ETC. 55 

phosphates normally present which come down when 
the reaction is made alkaline. Many urines produce 
a clear dark green solution when heated with the Peri- 
ling solution. This is a partial reduction reaction and 
like the other has no special importance as urines free 
from sugar give it. At other times urines free from 
sugar yield an almost colorless mixture when boiled 
with the Fehling solution. These peculiar reduction 
effects are due to the presence of uric acid, creatin, 
creatinin, pyrocatechin and several other substances 
and are generally characterized by discharge of the 
deep blue color of the solution without precipitation 
of the copper suboxide. Certain substances taken as 
remedies give rise to products in the urine which ex- 
ert a similar action. Occasionally, however, the 
amount of uric acid is so large that the reduction is 
accompanied by actual precipitation of the copper as 
red oxide. This fact is of interest as it makes the test, 
at times, somewhat uncertain, but it is a very simple 
matter to determine whether or not a great excess of 
uric acid is present, as will be pointed out later. The 
liability to error in the Trommer test from these causes 
is less than in the Fehling test, but notwithstanding this 
the latter must still be regarded as the better test prac- 
tically, because of its great convenience and the sharp- 
ness of the reaction with even traces of sugar. The 
ingredients of the Fehling test are best kept in sepa- 
rate bottles closed with rubber stoppers. A very con- 
venient arrangement is explained in the following 
paragraph. 

Two bottles, each holding about 200 cc, are fitted 



56 URINE ANALYSIS 

with perforated rubber stoppers. Through the open- 
ing in each stopper the stem of a 2 cc. pipette with 
very short tip is passed, and left in such a position 
that when the bottles are half filled the bulbs 
and stems to the mark will be covered with the liquid. 
One bottle contains the standard copper sulphate solu- 
tion, the other the mixture of alkali and tartrate solu- 
tion. The rubber stoppers should be covered with 
vaseline so that they will permit the pipette stems to 
slide easily in the perforations, and also close the bot- 
tles perfectly. When the stoppers are inserted the pi- 
pettes should stand full to the mark, ready for use. 

On withdrawing the stoppers with forefinger closing 
the pipettes, exactly 2 cc. of each liquid can be taken 
out without delay, and on mixing in a test-tube yield 
the Fehling solution, fresh and ready for use, directly, 
or after dilution with distilled water, as thought neces- 
sary. As the solutions are used the pipette steins 
are pushed farther through the stoppers so as to leave 
the marks always at the surface of the liquids. The 
solutions may be kept in this manner for years, and 
their use is not attended with any inconvenience. 
The open ends of the pipette stems should be kept 
closed with small rubber caps, or a bit of soft paraffin 
wax. The mixed Fehling liquid does not keep well 
unless prepared with certain unusual precautions, and 
therefore several other single solutions have been sug- 
gested, as described in the next paragraph. 

Other Copper Solutions. — The original Fehling solu- 
tion has been modified in various wavs. Most of these 



THE TESTS FOR SUGAR, ACETONE, ETC. 57 

modifications consist in mere changes in the proportions 
of the ingredients dissolved. Two, however, may be 
considered as fundamentally different. 

Loewe (1870) recommended a solution made by dis- 
solving copper sulphate in water, adding solution of 
sodium hydroxide and then glycerol. For certain pur- 
poses copper hydroxide was found to possess advan- 
tages over the sulphate. The preparation of the Loewe 
solutions is described in the appendix. The claim 
was made by Loewe that the addition of glycerol pre- 
vents the spontaneous decomposition of the blue solu- 
tion, which may, therefore, be kept mixed. While 
this is not absolutely correct it is true that the glycerol 
solutions keep much better than the mixed tartrate- 
alkali copper solutions as usually made, and have, 
therefore, found favor with some physicians. 

Schmiedeberg (1886) described a solution contain- 
ing in one liter 34.63 grams of crystallized copper sul- 
phate, 16 grams of mannitol and 480 cc. of sodium hy- 
droxide solution of 1. 145 sp. gr. This solution is eas- 
ily prepared and has also the advantage of perma- 
nence. 

Both the Loewe and the Schmiedeberg solutions 
have suffered slight alterations, without, however, 
being improved. 

The second suggestion of Loewe, i. e. , to use copper 
hydroxide instead of sulphate, has not been generally 
followed, but it certainly has in some cases decided ad- 
vantages. The final reaction in all these tests is the 
same as with the Trommer or Fehling test. 



58 URINE ANALYSIS 

The Bismuth Test. — Bottger found (1856) that in 
presence of alkali, bismuth subnitrate is reduced to the 
metallic condition by the action of dextrose in hot so- 
lution. As a urine test he recommended to make it 
strongly alkaline with sodium carbonate, and then add 
a very small amount, what can be held on the point 
of a penknife, of the pure bismuth subnitrate. On 
boiling the mixture the insoluble bismuth compound, 
which settles to the bottom, turns dark if sugar is 
present. 

The test is at present carried out by adding to the 
urine in a test-tube an equal volume of 10 per cent, 
solution of sodium or potassium hydroxide, and then 
the subnitrate. Boiling gives the reaction as before. 
In absence of sugar (or albumin) the bismuth com- 
pound remains white. 

In performing this test only a very small amount of 
the subnitrate should be taken. This is absolutely 
necessary in the detection of traces of sugar. In this 
case the reduction is but slight, and not much black 
powder of bismuth or its oxide can be formed. If a 
great excess of the white subnitrate is taken it may be 
sufficient to completely obscure the reduction product. 
It is frequently well to use not more than four or five 
milligrams of the subnitrate. 

The black, precipitate formed was at one time sup- 
posed to be finely divided metallic bismuth. Later 
investigations seem to show that it consists essentially 
of lower oxides of bismuth. This test has certain ad- 
vantages over the copper tests. It is easily made, and 
with materials evervwhere obtainable in condition of 



THE TESTS FOR SUGAR, ACETONE, ETC. 59 

sufficient purity. Furthermore, the reaction is not 
given with uric acid, which it will be remembered 
may act on the Fehling solution if excessive. 

Albumin, however, interferes with the test, as it 
gives, also, a black precipitate when boiled with al- 
kali and the bismuth subnitrate. In this case the al- 
bumin gives up sulphur and forms bismuth sulphide. 

If albumin is present in a urine it should be coag- 
ulated and filtered out before trying the bismuth test. 
Bruecke recommends to coagulate by means of a solu- 
tion of potassium bismuth iodide, the excess of bis- 
muth serving to complete the sugar test. The reagent 
for this purpose is made by dissolving freshly precipi- 
tated bismuth subnitrate in a hot solution of potassium 
iodide by the aid of some hydrochloric acid. This is 
the solution previously recommended by Fron for the 
precipitation of alkaloids, and is made by dissolving 
7 grams of potassium iodide in 20 cc. of water to which 
after heating 1.5 grams of the bismuth subnitrate and 1 
cc. of pure strong hydrochloric acid are added. The 
mixture must be kept hot until all is dissolved, result- 
ing in an orange-red solution. 

This reagent precipitates albumin, but as it is ren- 
dered turbid by water the amount of acid necessary to 
prevent this for a given volume must be ascertained 
before it can be used with urine. This can be deter- 
mined by adding a little of it (a few drops) to some 
water in a test-tube, and then dilute hydrochloric acid 
until the precipitate just disappears. The test proper 
is then made by taking the same quantity of urine and 
adding the same amount of acid and the reagent. 



60 URINE ANALYSIS 

Albumin and other disturbing substances precipi- 
tate, and can be filtered off. The clear nitrate should 
not be made turbid by acid or the reagent. It is next 
made strongly alkaline with potassium or sodium hy- 
droxide and then boiled. In presence of sugar a black 
precipitate is formed as before. 

After adding the reagent it is necessary to wait sev- 
eral minutes for a possible precipitate to form and set- 
tle. The addition of alkali to the nitrate produces a 
bulky white precipitate of bismuth hydroxide which 
is readily reduced at the boiling temperature by sugar 
present. If only traces of sugar are present the boil- 
ing must be long-continued to obtain the black pre- 
cipitate. What was said above about the danger of 
obscuring this precipitate by the white bismuth com- 
pounds obtains also here. 

When only a small amount of sugar is suspected it 
is best to allow the bismuth hydroxide precipitate to 
partially settle, and then to pour off the supernatant 
alkaline urine with a little of it. In this manner the 
amount of the bismuth compound which finally enters 
into the test is so small that it should all be reduced 
by even a trace of sugar, on subsequent boiling. When 
carefully performed this modification of the original 
Bottger test is a practically good one. It is not as 
sensitive as the Fehling test but shows traces of sugar 
of clinical importance. 

Fallacies in the Reduction Tests. — It has been shown 
above that several bodies normally found-in urine are 
able to reduce the alkaline copper solutions. Some 



THE TESTS FOR SUGAR, ACETONE, ETC. 6 1 

of these interfere with the bismuth reactions also, but 
not to the same degree ; but attention must be called 
to another source of error which is very important. In 
warm weather it is often desirable to add something 
to urine to prevent its rapid decomposition, and sev- 
eral substances have been suggested for the purpose. 
The best known are chloral, chloroform, salicylic acid, 
phenol, and formaldehyde. Unfortunately all of these 
except phenol have rather a marked action on the 
copper solutions. As a preservative phenol is objec- 
tionable from other standpoints. Urine intended for 
sugar tests should be tested as soon as possible after 
collection, and no foreign substance should be added 
as a preservative. Neglect of this very simple and 
obvious precaution has caused many serious blunders, 
especially in the examination of urine from applicants 
for life insurance. 

The Phenylhydrazine Test. — In this test a reaction 
discovered a few years ago has been applied by v. 
Jaksch to the examination of urine. Add to about 
10 cc. of urine 0.2 gram of phenylhydrazine chloride 
and a slightly greater amount of sodium acetate. Warm 
the mixture gently, and if solution does not take place 
add half the volume of water and heat half an hour 
on the water-bath. Then cool the test-tube by pla- 
cing it in cold water and allow it to stand. If sugar is 
present a yellow precipitate settles out, which consists 
of minute needles generally arranged in rosettes, visi- 
ble under the microscope. Albumin does not obscure 
this test, but if much is present it is best to coagulate 



62 URINE ANALYSIS 

it as well as possible by boiling, and filter. The yel- 
low precipitate is called phenylglucosazone. 

For the detection of traces of sugar by this method 
it is necessary to use more urine and more of the re- 
agents. 50 cc. of urine with 2 grams of phenylhydra- 
zine chloride and 3 grams of the acetate may be taken. 

Phenylglucosazone melts at 205 ° C. and a determi- 
nation of the melting-point may be made as a confirma- 
tory test. For this purpose the supernatant liquid is 
poured off and the fine yellow crystals are washed 
with water by decantation. They are transferred to a 
small watch-glass, allowed to dry over sulphuric acid 
in a desiccator, and are then ready for the test. Melt- 
ing-points are usually found by placing a small amount 
of the substance in question in a thin narrow tube, 
which is fastened to a thermometer by means of rub- 
ber bands. The substance in the bottom of the tube 
must be near the bulb of the thermometer. The bulb 
and bottom of tube are then immersed in a beaker of 
oil or sulphuric acid which is gradually heated until 
the substance begins to fuse. The temperature indi- 
cated by the thermometer is taken as the melting- 
point. For best methods of working this test of find- 
ing the fusing-point some standard manual of organic 
chemistry should be consulted. 

On the whole, it must be said that this reaction is 
of very limited applicability in urine analysis. It has 
value only when the copper or bismuth methods are 
insufficient to decide concerning the presence or ab- 
sence of sugar. In cases having real clinical impor- 
tance such uncertaintv is rare. 



THE TESTS FOR SUGAR, ACETONE, ETC. 63 

The a-naphthol Test. — This depends on the reaction 
between a-naphthol and sugar in presence of sulphuric 
acid, and was discovered by Molisch. Take about a 
cubic centimeter of urine, previously diluted with five 
to ten volumes of water, and add to it two drops of a 
20 per cent, solution of a-naphthol in alcohol. Then 
add about half a cubic centimeter of strong sulphuric 
acid and agitate. A blue color indicates sugar. If 
the acid is carefully added so as to flow under the 
lighter liquid, a blue zone is formed between them. 
By diluting largely with water and shaking, a violet 
precipitate is produced. 

This method is exceedingly delicate, but unfortu- 
nately is not characteristic, as many substances show 
the same result. The trace of sugar, or similar body, 
normally present, gives a marked reaction; hence the 
direction to largely dilute the urine before adding the 
reagent. 

The a-naphthol may be replaced in this test by a 
20 per cent alcoholic solution of thymol. The mixture 
becomes dark red and carmine-red on dilution with 
water. It has been shown that these color changes 
depend on the formation of small amounts of furfural 
by action of sulphuric acid on traces of carbohydrates 
and the subsequent combination of the furfural with 
the a-naphthol or thymol. However, not only carbo- 
hydrates, but also albumins and many other substances 
yield furfural in this manner and in normal urine some 
of these substances may be always present. 

Molisch claims that the reaction found with highly 
diluted urine is a sugar reaction, that in condition of 



64 URINE ANALYSIS 

high dilution other bodies which may possibly be 
present cannot give this test. A color still shows 
when normal urine diluted ioo times is used, and on 
this behavior, partly, the claim that sugar is normally 
always present in urine is made. It will be seen from 
this that the test is too sensitive for ordinary clinical 
needs, but as a laboratory test it is valuable. By 
attentive study of the behavior of diluted normal and 
diabetic urines the chemist soon learns to recognize 
the deeper colors obtained with the latter and is there- 
fore able to employ the test in the way of confirma- 
tion. 

The Fermentation Test. — When yeast is added to 
urine containing sugar and the mixture left in a mod- 
erately warm place the usual fermentation soon begins 
which is shown by two principal changes. Carbon 
dioxide is given off, which may be collected and iden- 
tified, and the mixture becomes lighter in specific 
gravity. When only traces of sugar are present the 
test by collection and identification of the carbon di- 
oxide frequently fails because of the solubility of the 
gas in the liquid. 

The variation in the specific gravity is an indication 
of greater value, as it can be readily observed with 
proper appliances. The test has practical value, how- 
ever, only as a confirmation of some other one. If by 
the copper solutions, for instance, a strong indication 
is obtained which it is suspected may be due to an 
excess of uric acid, the reaction by fermentation may 
be resorted to because only sugar will respond to it. 



THE TESTS FOR SUGAR, ACETONE, ETC. 65 

The test may be made by pouring 100 cc. of the urine 
into each of two bottles or flasks. To one, half a cake 
of compressed yeast, crumbled, is added; the other is 
left pure. The bottle with the yeast is closed by 
means of a perforated stopper (to allow escape of gas), 
while the other is tightly corked. The two are left 
side by side, in a warm place about twenty-four hours. 
At the end of this time a test of the specific gravity of 
the contents of both bottles is made. If sugar is pres- 
ent to the amount of one-half per cent, the specific 
gravity of the yeast-bottle should be perceptibly lower. 
The test is frequently recommended as a quantita- 
tive one, as there is a fairly definite relation between 
amount of sugar and loss in density. 

Other Sugar Reactions. — Many other tests have been 
proposed for the detection of sugar in urine. A few 
of these will be referred to briefly in this place. 

One of these, the picric acid test, is based on the 
fact that a urine containing sugar when mixed with 
solutions of potassium hydroxide and picric acid and 
boiled, turns a dark mahogany-red from formation of 
picramic acid. 

When urine is made strongly alkaline with potas- 
sium hydroxide and treated with a weak solution of 
diazobenzene sulphonic acid in water, it turns reddish 
yellow if sugar is present, and becomes afterward 
claret-red and finally dark red if much is in solution. 
The reaction is delicate, but is given by other bodies 
than sugar. 

Another test depends on the reaction between sugar 
5 



66 URINE ANALYSIS 

solutions and indigo-carmine in presence of alkali. 
The urine is made alkaline with sodium carbonate and 
treated with indigo-carmine until a deep blue is ob- 
tained on heating. If sugar is present, on longer heat- 
ing the color fades to yellow by reduction. The color 
returns bv cooling: and shaking with air. 

Weak aqueous solutions of methylene-blue are de- 
colorized when boiled with alkaline dextrose solutions. 
To apply this in urine analysis a solution of the methy- 
lene-blue is made by dissolving about 30 milligrams 
in 100 cc. of water. In a test-tube take 5 or 6 cc. of 
this solution, add 2 cc. of a 5 per cent, potassium hy- 
droxide solution and then the diluted urine. On boil- 
ing, the color fades or disappears completely if much 
sugar is present. In urines containing much sugar a 
reaction is given distinctly with 2 cc. after tenfold 
dilution. 

A very characteristic reaction is given by the aid of 
the coloring-matter known as safranine. This occurs 
in commerce as a mixture of three related bodies bav- 
ins; the empirical formulas, C H N CI, C H N CI, 

<=> i ' 21 21 4 ' 20 19 4 > 

and C H N CI. It is soluble in water with a red 
19 17 4 

color and a solution of 1 part to 1000 of water is em- 
ployed as the reagent. A 5 per cent, potassium hy- 
droxide solution is also used and the test is made by 
adding to a small volume of the suspected urine an 
equal volume of the alkali solution and then the safra- 
nine solution in like amount. About 3 cc. of each 
should be taken, and the mixture then boiled. In pres- 
ence of sugar to the extent of one-tenth per cent, or 
more the mixture is decolorized, a pale yellow result- 



TESTS FOR SUGAR, ACETONE, ETC. 67 

ing. With stronger sugar urines more of the safranine 
solution is decolorized and a rough measure of the 
amount of sugar present may be made by noting the 
volume of safranine solution in which the color is de- 
stroyed by the original urine taken. If the test-tube 
in which the test is made be allowed to stand exposed 
to the air the red color will return soon in the upper 
part of the liquid by oxidation. The loss of color in 
the mixture depends on the reduction of the safranine ; 
fortunately but few substances likely to be present in 
urine show the same behavior, and on the whole 
the test may be considered the best of the color reac- 
tions. It has been very highly recommended and may 
be used when there is doubt concerning the copper or 
bismuth indications. 

The Amount of Sugar 

It is not always sufficient to be able to detect the 
presence of sugar in urine. A knowledge of the amount 
is frequently of the greatest importance. A number 
of methods have been proposed by which a quantita- 
tive determination can be made, some of them crude 
and of little practical value, while others give, when 
properly carried out, results which are accurate. The 
methods in general may be divided into four groups, 
depending on the 

(1) Reduction of solutions of heavy metals, and 
measurement of the amount of reduction. 

(2) Change of color produced in organic solutions, 
by action of sugar, the depth of final color be- 
ing proportional to the amount of sugar. 



68 URINE ANALYSIS 

(3) Results of fermentation with measurement of 
change in specific gravity of the urine or meas- 
urement of evolved carbon dioxide. 

(4) Observation of rotary polarization of light. 

Methods by Reduction of Metallic Solutions 

The reduction methods are illustrated in the use of 
the Fehling solution as a qualitative test and in the 
bismuth tests. The general principles involved in 
making a quantitative determination of sugar by aid 
of the Fehling solution are the same as those involved 
in making other volumetric analyses with standard 
solutions, and are fully explained in the author's work 
on chemical physiology, or in any one of the standard 
manuals of volumetric analysis in general use. A 
measured volume of the properly prepared Fehling 
solution is poured into a flask and brought to the boii- 
ing-point. Then from a burette the dilute sugar solu- 
tion is run in slowly, a few cubic centimeters at a 
time, until the deep blue of the copper solution is just 
discharged, leaving a pale yellow. At this stage the 
copper is all reduced to the condition of insoluble red 
suboxide, Cu O. If the Fehling; solution is used in 

'2 O 

undiluted condition and the sugar is present in solu- 
tion of about 1 per cent, strength, then each cubic cen- 
timeter of the Fehling solution is reduced by 4.75 
milligrams of dextrose. If the Fehling solution is 
diluted with four volumes of water before use its oxi- 
dizing power is slightly greater, each cubic centimeter 
being equivalent to 4^94 milligrams of dextrose; 25 or 
50 cc. of the Fehling solution should be taken and, if 



TESTS FOR SUGAR, ACETONE, ETC. 69 

undiluted, the 118.75 or 237.5 milligrams of sugar re- 
quired for reduction must be contained in the volume 
of solution added from the burette. A simple calcula- 
tion gives the strength of the saccharine liquid in 
grams per liter, or in per cent, by weight if the spe- 
cific gravity is known. 

When applied to the urine, however, the process 
requires certain modifications because of the fact that 
this secretion contains always a number of substances 
which interfere to some extent with the normal reduc- 
tion and precipitation of the copper suboxide. The 
•determination of dextrose in aqueous solution by the 
Fehling liquid is a problem of extreme simplicity, but 
in urine the case is somewhat different. 

If we measure out 50 cc. of the mixed Fehling so- 
lution, heat it to boiling and then run in the saccha- 
rine urine from a burette, it frequently happens that a 
greenish yellow muddy precipitate forms which does 
not turn bright red and which, instead of quickly set- 
tling to the bottom of the flask, remains suspended 
and makes it impossible to observe the disappearance 
of the blue color indicating the end of the reduction. 
This difficulty may be largely obviated by working 
with solutions of greater dilution, as explained in the 
following paragraph. 

Determination by Fehling Solution. — Prepare a Fehl- 
ing solution as shown in the appendix and then accu- 
rately mix it with four volumes of distilled water, that 
is, to 100 cc. add 400 cc. of water, to 50 add '200, or 
to 25 add 100. In any event the dilution must be 
accurately made. One cubic centimeter of this liquid 



70 URINE ANALYSIS 

will oxidize almost exactly one milligram of dextrose 
as shown above, provided the sugar is in approximately 
i per cent, solution. For all practical purposes of 
urine analysis the oxidizing power may be considered 
the same in a solution of one-half per cent, strength, 
and only very slightly increased in still weaker solu- 
tions. Therefore, before beginning the test dilute the 
urine, if necessary, accurately with four or nine vol- 
umes of water. This can be done by making 50 cc. up 
to 250 or to 500 cc. and mixing well by shaking. 

With the diluted urine so prepared proceed as fol- 
lows : Measure out 50 cc. of the dilute Fehliug solu- 
tion, pour it in a flask and heat to boiling on gauze. 
Fill a burette with the diluted urine and when the 
solution in the flask is actively boiling run in about 
3 cc. Boil two minutes, remove the lamp, and wait 
half a minute to observe the color. If blue is still 
visible, heat to boiling again and run in 3 cc. more. 
After boiling two minutes as before, wait a short time 
and observe the color near the surface of the liquid in 
the flask. If still blue repeat these operations until 
on waiting it is found that the blue has given place 
to a yellow. The urine should be so dilute that at 
least 10 cc. must be run in to reduce all the copper 
hydroxide. 

When the volume required is found to within 2 or 
3 cc. a second experiment must be made, the urine 
being added very gradually now, without interrupting 
the boiling longer than necessary, until the first of the 
limits between which the correct result must lie, as 
shown by the former test, is reached. From this point 



TESTS FOR SUGAR, ACETONE, ETC. 7 1 

the addition of the urine is continued, with frequent 
pauses for observation of color, until the reduction is 
complete. The volume of urine used contains 50 mil- 
ligrams of sugar. 

If the preliminary experiment shows that the urine 
is strong in sugar and that the reduction is easy, that 
is, that the cuprous oxide separates and settles readily, 
the second test may advantageously be made with 50 
cc. of a stronger Fehling solution. With many strong 
diabetic urines it is possible to use the undiluted cop- 
per solution with the oxidizing power of 4.75 milli- 
grams of sugar to each cubic centimeter. The diffi- 
culties in this test have been very much overestimated; 
with a little practice any one can make a good sugar 
determination in urine. The important point is to 
find by a few simple preliminary tests the best condi- 
tions of dilution of Fehling solution and urine to give 
a precipitate which settles readily. With this infor- 
mation, and it can be acquired in a few minutes, the 
actual quantitative experiment can be easily made. 

As an illustration of the calculations involved let it 
be assumed that 50 cc. of the dilute Fehling solution 
is reduced by 1 1 cc. of urine. Each cubic centimeter 
of the urine must therefore contain 4.54 milligrams of 
sugar. If the urine were undiluted this corresponds 
to 4.54 grams to the liter. If it had been diluted with 
9 volumes of water the result must be multiplied by 
10, giving as the original strength 45.4 grams per 
liter. If the specific gravity of the urine were found 
to be 1.032, the percentage strength would be 

-^ = 4.39. 
1.032 4dv 



72 URINE ANALYSIS 

The Use of Pavy's Solution. — To avoid some of the 
difficulties in the titration of diabetic urine by the 
Fehling solution, Pavy suggested a solution contain- 
ing ammonia. If a solution of dextrose is run into a 
boiling copper solution containing ammonia in con- 
siderable quantity the copper is gradually reduced, 
giving finally a clear, colorless solution instead of a 
red precipitate. The end of the reduction is, there- 
fore, indicated by disappearance of color alone. The 
preparation of the Pavy solution is given in the ap- 
pendix. Its strength as there described is just one- 
tenth of that of the common Fehling liquid ; that is, 
ioo cc. oxidizes 50 milligrams of dextrose. The test 
is performed in a flask, as is the Fehling titration ; but 
as the solution is easily changed by atmospheric oxi- 
dation, just as soon as it begins to hold some reduced 
copper, precautions should be taken to exclude the air 
during titration. This can be done by passing a slow 
'current of illuminating gas or hydrogen through the 
flask during the test, or, better, by adding to the con- 
tents of the flask enough white paraffin wax to form, 
on melting, a protective liquid layer several millime- 
ters in depth. 

The titration is carried out as follows: Measure 100 
cc. of the ammoniacal copper solution into a flask hold- 
ing about 300 cc. Throw in some small pieces of 
pumice stone to prevent " bumping," and then heat 
to the boiling-point on wire gauze. The sugar solu- 
tion must be dilute, and should be contained in a bu- 
rette with a delivery tip bent to one side and then 
down, so that the contents of the burette can be added 



TESTS FOR SUGAR, ACETONE, ETC. 73 

slowly but continuously to the liquid in the flask without 
interrupting the ebullition. The operation should be 
carried out where there is a good circulation of the air 
to carry off the evolved ammoniacal fumes. As 
the reduction is very slow the addition of the sugar 
solution must not be rapid. There is danger of add- 
ing too much until the operator becomes familiar 
with the method. If the precautions mentioned are 
neglected, which is usually the case, the results come 
out a little too low, because the air reoxidizes some of 
the ammoniacal cuprous solution, making it necessary 
to add more of the sugar to complete the reduction ; 
that is, to completely discharge the color. 

The method yields at best only approximate results, 
and working it subjects the analyst to the annoyance 
of ammoniacal fumes, unless the apparatus is compli- 
cated by the addition of a delivery tube to carry the 
ammonia through a window or into a fume chamber. 

The reducing power of the copper in this solution 
depends, to some extent, on the amount of ammonia 
present, and from the fact that this is lost by ebulli- 
tion during the performance of the test, irregularly 
and at different rates in different experiments, it fol- 
lows that the results obtained cannot be perfectly uni- 
form. Besides this the solution does not keep per- 
fectly, its reducing power slowly undergoing change. 
However, the method has value and should be learned, 
because it can be rapidly worked and the results ob- 
tained are sufficiently accurate for clinical purposes. 

The solution has been still further modified by sub- 
stituting glycerol for the tartrate, giving what may be 



74 URINE ANALYSIS 

called the Loewe-Pavy solution. This solution is em- 
ployed as is the Pavy liquid and has the same advan- 
tages and drawbacks. For method of preparation see 
the appendix. It is claimed for it, however, that it 
keeps somewhat better. Solutions containing ammo- 
nia cannot be used for qualitative testing. 

Sugar Test by Solutions of Mercury. — Certain solu- 
tions of mercury, like those of copper and bismuth, 
are readily reduced by alkaline dextrose solutions and 
may be employed in titration. Two such solutions 
are frequently used; £7>.,Knapp's solution, containing 
mercuric-potassium cyanide, and Sachsse's solution, 
containing mercuric-potassium iodide. See the ap- 
pendix for the preparation of both of these. 

The solution of Knapp is frequently used in urine 
titration and is employed in the following manner : 10 
cc. of the solution, corresponding to 25 milligrams of 
dextrose, are diluted with 25 cc. of water in a flask and 
heated to boiling. The urine, which has been pre- 
viously diluted accurately with from four to nine vol- 
umes of w T ater, is run from a burette into the hot liquid 
until the whole of the mercury is precipitated, which 
can be recognized as follows : Allow the precipitate to 
settle and then by means of a glass rod place a drop of 
the yellowish supernatant liquid on a piece of white 
Swedish filter-paper. Hold the paper then over an 
open hydrochloric acid bottle containing the fuming 
acid, and afterward over a beaker containing some 
strong hydrogen sulphide water. If the drop of trans- 
ferred liquid contains even a trace of mercury this will 
be shown by the formation of a brown stain. In this 



TESTS FOR SUGAR, ACETONE, ETC. 75 

case it will be necessary to add more of the sugar so- 
lution, and repeat the operations until the complete 
reduction and precipitation of the mercury compound 
is accomplished, as shown by a negative result with the 
hydrogen sulphide test. 

This method has been found to give very excellent 
results, but longer practice is necessary to give pro- 
ficiency with it than with the other. Besides the 
method given above, others have been suggested for 
the determination of the end of the reduction, but they 
do not give exactly the same value for the oxidizing 
power of the mercuric solution. 

Color and Fermentation Methods 

The methods of quantitative sugar analysis depend- 
ing on comparison of colors in sugar solutions acted 
on by picric acid and alkali or other reagent, are neither 
very convenient nor accurate. 

The fermentation test is sometimes applied quanti- 
tatively, but in those cases where it is the most accu- 
rate it is least necessary. With very weak sugar so- 
lutions it can only be used with the most careful re- 
gard to changes in temperature by the method referred 
to above. With strong diabetic urines accurate re- 
sults are more readily reached, but here, by dilution, 
the copper solutions give the desired information more 
quickly and accurately. 

When the saccharine urine is fermented as described 
and a change of specific gravity observed, the percent- 
age of sugar is approximately given by multiplying 
each 0.00 1 lost by 0.23. For instance, if the urine be- 



76 URINE ANALYSIS 

fore fermentation had a specific gravity of 1.032, and 
after fermentation, at the same temperature, a specific 
gravity of 1.016, we have 16 X 0.23 = 3.68 as the per 
cent, of sugar present. 

Sugar Determination by Polarimetry 

The construction and method of using the polar- 
imeter are fully explained in the author's work on 
"Chemical Physiology," to which the reader is re- 
ferred. In practical work several forms of polariza- 
tion apparatus are in actual use, but those known as 
"half shadow" instruments must be considered the 
most convenient and generally applicable. 

The direct examination of urine is not always pos- 
sible because of its color, and sometimes because of its 
slight turbidity. The best results are obtained with 
colorless and clear solutions. It is therefore sometimes 
necessary to prepare the urine by a preliminary treatment 
before it can be filled into the observation tubes. Dia- 
betic urines light in color ma}- frequently be used 
after simple filtration to render them perfectly clear, 
especially with the high-class modern instruments of 
the half-shadow type with which a good illumination 
can be secured. If the urine is much colored, so that 
an observation cannot be made with the shortest tube 
— 100 millimeters in length — which can be deter- 
mined by a simple trial, resort must be had to pre- 
cipitation to remove part of the color. Several pre- 
cipitating agents are used for clarifying sugar solu- 
tions for the polariscope. The simplest of these is a 
solution of basic lead acetate which produces a volu- 



TESTS FOR SUGAR, ACETONE, ETC. 77 

minons precipitate that carries down much coloring- 
matter. This is frequently used alone, but perhaps 
better combined with alum. When the basic acetate 
is added first and then some aluminum sulphate the 
mixed precipitate is flocculent and very effective in 
carrying down coloring-matters. Use the basic ace- 
tate of lead described in the appendix and prepare a 
solution of aluminum sulphate of about equivalent 
strength ; that is, of such strength that 1 cc. will pre- 
cipitate the lead of 1 cc. of the other. 

Measure out 100 cc. of the urine, add 5 cc. of the 
lead solution and 2 or 3 cc. of the alum solution, shake 
well, add water to bring the volume to nocc. exactly, 
shake again and allow to stand ten minutes. Then 
filter through a dry filter. The filtrate will be found 
much lighter in color than the original and probably 
suitable for use. If it is opalescent pour it through 
the precipitate on the filter when it will be found 
much brighter. 

There is a slight loss of sugar in this operation as 
some is carried down by the precipitate. The clari- 
fied solution is then filled into the polarization tube 
and observed in the usual manner. The result ob- 
tained must be increased by one-tenth because of the 
dilution of the original urine. As the precipitate 
formed occupies an appreciable volume when dried, 
the clarified solution is correspondingly concentrated 
and the reading from this cause w T ould be too high. 
For our purpose, however, we can assume that the 
gain in concentration is counterbalanced by the loss 
of sugar inclosed with the precipitate and neglect both 
sources of error. 



78 URINE ANALYSIS 

If the urine contains albumin it must be separated 
by coagulation and filtered out, because it rotates the 
plane of polarized light to the left, and would there- 
fore make the amount of sugar appear lower. 

A given volume of urine is poured into a beaker and 
enough dilute acetic acid is added to give a faint re- 
action ; it is then boiled, and after standing five min- 
utes filtered. As all albuminous bodies, however, are 
not precipitated by simple coagulation with acetic 
acid, it has been recommended to add to ioo cc. of the 
urine, 10 cc. of the strongly acid solution of phospho- 
tungstic acid, already described, and filter after ten 
minutes. The dilution must be allowed for in the 
final calculation. Some coloring-matters are also re- 
moved by this treatment. The urine may contain 
other active substances, but in amount so small that 
their effect may be neglected entirely. 

Calculation of Result. — For sodium light the formula 

fa] = I^- tt 
L J Ic 

is used with the factor [a] =52.7°. 
In this formula [a] = the so-called "specific rotation" 
of the sugar, which is a constant and must be assumed 
as known, cl is the angle of rotation observed in the 
actual test, / is the length of the observation tube ex- 
pressed in decimeters, while r, finally, is the strength 
of the solution, expressed in grams per 100 cc, desired. 
As we observe a directly we may write, 

iooa 

c ~ ~i^T° • 



TESTS FOR SUGAR, ACETONE, ETC. 79 

that is, the number of grams of diabetic sugar in 
100 cc. of the solution polarized is equal to the prod- 
uct of the observed angle of rotation multiplied by 
100 and divided by the product of the length of the 
observation tube in decimeters multiplied by the spe- 
cific rotation, 52. 7 . 

If in a given case we find a rotation of io° 32.4', 
with a tube two decimeters long, our formula becomes 
100 X 10.54 
c= 2X52.7° =I ° ; 
that is, the concentration, c, is 10 grams per 100 cc. 

With a decimeter tube each degree of rotation cor- 
responds to a concentration of 1.8976. With the usual 
two-decimeter tube each degree indicates 0.9488 gram 
in each 100 cc. 

The specific rotation of dextrose as obtained from 
urine appears to be a little higher than is that of the 
product made from starch. 

Other Sugars in Urine 

Pathologically, traces or even larger quantities of 
several other saccharine bodies are occasionally found 
in urine. Among these we have first : 

Levulose, or Fruit Sugar. — This is found along with 
dextrose in some cases of diabetes, but does not appear 
to occur alone. 

While the recognition of levulose in the pure state 
or in simple aqueous solution is a matter presenting 
no difficulty, the certain detection of this body as it 
occurs in urine is by no means as readily effected. This 
sugar gives the reduction and fermentation tests as 



So URINE ANALYSIS 

described under dextrose, and therefore cannot be dis- 
tinguished by these methods. Levulose, however, 
rotates the plane of polarized light to the left, and 
this property is sometimes of service in aiding the 
recognition. If the rotation is strongly to the left, 
the presence of levulose in quantity may be inferred, 
assuming that albumins are absent. If the quantity 
of sugar, calculated as dextrose, determined by polari- 
zation, is much below that found by the copper reduc- 
tion method, the indication is that levulose is present 
with the dextrose. An exact measurement of the 
amounts of the two sugars when mixed in the urine 
is not possible with present means. 

Lactose, or Milk-sugar, is occasionally found in the 
urine of nursing women. Its certain detection when 
in small amount presents even greater difficulties than 
is the case with levulose. As its rotation is right- 
handed the polariscopic test is of little value. 

Milk-sugar is more strongly acted on by Fehling 
solution than is dextrose. While i cc. of the copper 
solution oxidizes 4.75 milligrams of dextrose, it oxi- 
dizes 6.76 milligrams of milk-sugar. 

When a solution of milk-sugar is boiled with dilute 
hydrochloric acid it yields dextrose and galactose, the 
latter resembling dextrose in its behavior with the 
copper solution. The specific rotation [a] D , of dex- 
trose is 52. 7 , of lactose, 52. 5 , while that of galactose 
is 81. 3 . The specific rotation of a mixture of equal 
parts of dextrose and galactose has been found by ex- 
periment to be 67. 5 , which agrees closely with the 



TESTS FOR SUGAR, ACETONE, ETC. 8 1 

mean of 52. 7 ° and 81. 3 . If, therefore, the rotation 
of urine is increased after heating with acid and neu- 
tralizing, and its copper reducing power also increased, 
we have data suggesting the presence of milk-sugar. 
Experiments to show these points with certainty must 
be very carefully conducted, consuming no little time 
in manipulation. They are, therefore, of little value 
from a clinical standpoint. 

Inosite, or Muscle-sugar, has been found in urine, in 
diabetes, and also with albumin. There is no simple 
method by which it may be separated in the small 
quantity in which it occurs in urine. 

Other Carbohydrates. — Traces of a body resembling 
erythrodextrin have been reported as occurring some- 
times in ■ urine along with sugar, or after disappear- 
ance of the same. But the origin and clinical signifi- 
cance of this substance are so obscure that no further 
attention need be given it here. The same is true 
of the so-called animal gum referred to by many 
writers. It appears to be found in small amount in 
all urines, and may be separated by several methods. 
If present in relatively large quantity, it may be recog- 
nized by boiling the urine first with dilute sulphuric 
acid, then neutralizing and testing with Fehling solu- 
tion. By the acid treatment it is converted into a 
sugar-like substance which reduces alkaline copper so- 
lutions. 

Acetone 

This is a substance which frequently is found in 
urine in small amounts. Indeed, it may be true, as 

6 



82 URINE ANALYSIS 

has been asserted, that it is normally always present 
in traces. This physiological acetonnria has no clini- 
cal significance. Under some circumstances, how- 
ever, it may be found in larger quantity, sometimes in 
amount sufficient to be detected by the odor alone, 
which fact first called attention to it. At one time it 
was supposed to be related to the sugar found in urine, 
but it is now established that it more generally accom- 
panies albumin and is frequently observed in many 
febrile conditions. 

Acetone in urine is believed to be a decomposition 
product of albumins, or of bodies which ma}- in turn 
be looked upon as resulting from proteid disintegra- 
tion, as will be pointed out below. It has been shown 
that in health, even, it can be much increased by a 
diet rich in nitrogenous materials. 

But, occurring as it does in fevers and in advanced 
stages of diabetes mellitus, a certain interest attaches 
to its detection, and numerous methods have been pro- 
posed by which it may be identified in small amount. 
Those which depend on its direct recognition in the 
urine are mostly uncertain. It is always safer to dis- 
til the liquid and apply the test to a portion of the 
distillate. Half a liter, or more, of the urine is poured 
in a retort attached to a Liebig's condenser, and, after 
addition of a little phosphoric acid, is subjected to dis- 
tillation, ioo cc. of distillate will be enough. A por- 
tion of this can be taken for each test as follows : 

LegaPs Test. — Add to 25 cc. of the liquid a small 
amount of a fresh solution of sodium nitroprusside, 



TESTS FOR SUGAR, ACETONE, ETC. 83 

and a few drops of a 50 per cent, potassium hydroxide 
solution. If a ruby-red color appears which slowly 
gives place to yellow, and if the addition of acetic acid 
changes this to purple, or violet-red, the presence of 
acetone is indicated. 

Lieben's Test. — This depends on the production of 
iodoform, and is carried out in this manner. To about 
5 cc. of the distillate add a few drops of a solution of 
iodine in potassium iodide (the "compound solution 
of iodine," Lugol's solution), and then a small amount 
of potassium hydroxide, to marked alkaline reaction. 
If acetone is present a yellowish white precipitate soon 
appears, which, on standing, becomes crystalline and 
more deeply colored. The test is said to be sharper 
and more characteristic if ammonia is used instead of 
the fixed alkali. The liquid is first made strongly 
alkaline with ammonia, and then the iodine solution 
is added until the brownish precipitate formed at first 
dissolves very slowly. In a short time the yellowish 
iodoform precipitate makes its appearance. A rough 
quantitative measure of the amount of acetone present 
is given by noting the smallest volume of the distil- 
late with which a distinct iodoform reaction can be 
seen. It is said that 0.0001 milligram in 1 cc. can be 
detected. 0.5 milligram in 10 cc. can be recognized 
by the nitroprusside reaction. 

Creatinin gives a ruby-red color as does acetone 
when the nitroprusside reaction is directly applied to 
urine, but after adding acetic acid a green or blue 
color results. 



84 URINE ANALYSIS 

Chautard's Test. — This depends on the production 
of a violet color in a solution of rosaniline hydrochlo- 
ride decolorized by sulphurous acid. To prepare the 
reagent dissolve i gram of the rosaniline salt (aniline- 
red, fuchsin, magenta) in a liter of water. Into this 
solution a current of sulphurous acid gas is led until 
the red color is destroyed, or converted to yellow. A 
great excess of the sulphurous oxide must not be used. 
A strong solution of the acid may be used in place of 
the gas. To 25 cc. of this reagent add an equal amount 
of the acetone distillate. In presence of the latter body 
a reddish violet color is produced. A similar reaction 
is given by aldehydes and ketones in general, but in 
a distillate from urine acetone is indicated with prac- 
tical certainty. One part of acetone in 1000 parts of 
distillate may be recognized by the test. 

Acetone has the composition C H 6 0, or CH .CO.CH , 
dimethyl ketone. It may be readily derived from the 
substance to be next described, acetoacetic acid, which 

has the formula CH CO.CH .CO H. This bv distilla- 

322 •> 

tion with acid yields acetone and carbonic acid. 

Acetoacetic or Diacetic Acid 

This compound is very frequently found associated 
with acetone in the urine of fevers and in diabetes 
mellitus. While acetone may occur in very small 
amount normally it is believed that acetoacetic acid 
is ahvays pathological. In the past few years much 
has been written on the subject of this substance and 
its clinical significance. It appears from the discus- 
sion that its presence in diabetes is of especial impor- 
tance and that anv increase in its amount should be 



TESTS FOR SUGAR, ACETONE, ETC. 85 

carefully followed by analytical tests. What is known 
as the coma of diabetes is closely associated, according 
to eminent authority, with the presence of acetoacetic 
acid in the blood. 

As to the origin of this acid body there has been 
much speculation, and at the present time the prob- 
lem is far from solution. At one time the presence of 
the acid was supposed to be dependent on that of 
/3-oxybutyric acid, the two bodies being usually asso- 
ciated. But the diacetic acid has been found in urine 
free from the other, and the assumed relation does not 
appear, therefore, to exist. While often present in 
large amount, relatively, in advanced stages of diabetes 
the relationship of this body to the excreted sugar has 
never been clearly established. 

Inasmuch as this acid is but slightly stable it need 
be looked for only in comparatively fresh urine. As 
it yields acetone in decomposition the tests for this 
body should be made first. If they show a negative 
result it is useless to go farther. But if positive the 
diacetic acid may be looked for. 

Ferric Chloride Test. — Our main test for acetoacetic 
acid depends on a reaction with ferric chloride with 
which it strikes a red color. Normally, there is noth- 
ing in urine which gives the same reaction, so that, if 
on the addition of a few drops of solution of ferric 
chloride to fresh urine a wine-red color results, the 
presence of acetoacetic acid may be inferred. At the 
present time, however, many coal-tar products are given 
as remedies which yield compounds that, on elimina- 
tion with the urine, give a red or purple color with 



86 URINE ANALYSIS 

ferric chloride when added. To detect the acetoacetic 
acid with certainty under these conditions it is neces- 
sary to proceed with greater care. To this end add to 
the urine, which should be fresh, a few drops of ferric 
chloride or enough to precipitate the phosphates pres- 
ent. Filter and add a little more of the chloride. A red 
color indicates the acid. Divide the liquid into two 
portions ; boil one and allow the other to stand a day or 
more. In the boiled portion the color due to acetoacetic 
acid should disappear within a few minutes, while in 
the other it should remain about twenty-four hours. 
Acidulate another portion of the urine with dilute 
sulphuric acid and extract it with ether which takes 
up acetoacetic acid. Remove the ethereal layer and 
shake it with a very dilute aqueous solution of ferric 
chloride. The red color in the new aqueous layer should 
appear as before and disappear on boiling, which be- 
havior distinguishes the acid from other substances 
likely to be present. 

B-Oxybutyric Acid. — The detection of this acid in 
the urine by chemical methods is by no means simple, 
but if present in amounts not too minute it may be 
found by the aid of the polariscope, inasmuch as its 
solutions possess a strong negative rotation. In di- 
lute solution the specific rotation is approximately 
[a] D = — 23. 4 . As this acid is found associated 
with sugar in diabetes it is necessary to destroy the 
sugar by fermentation before making the test. Amounts 
as high as 200 grams in the day's urine have been re- 
ported, but usually, where present at all, the amount is 
far below this, 15 to 20 grams being nearer the average. 



Chapter IV 

THE COLORING=riATTERS IN URINE 
BILIARY ACIDS 

Normal Coloring matters 

Although many investigations have been carried 
out on the subject of the normal urinary pigments we 
are yet unable to give a very definite account concern- 
ing them. This is partly due to the fact that the col- 
oring substances exist in the urine in minute traces 
only, which makes their separation and recognition 
exceedingly difficult, and partly to another fact that 
some of them are easily altered or destroyed by the 
action of the reagents employed in their investigation. 
By proceeding according to different methods, physi- 
ologists have obtained very different results indicating 
the existence of several colors, or at any rate modifi- 
cations of colors. The difficulty of detecting the nor- 
mal colors in urine is sometimes increased by the pres- 
ence of traces of accidental coloring-matters having 
their origin in peculiar or unusual articles of food con- 
sumed. Some of these will be referred to below. 

It seems to be settled, however, that in health not 
merely one but several coloring-bodies must be pres- 
ent. It has not yet been found possible to separate 
these in the free state. 



88 URINE ANALYSIS 

Uroerythrin is the name given by Thudichtim and 
others to a common reddish coloring-matter which 
often precipitates with urates and other substances. 
It is colored green by solution of potassium hydroxide, 
but the color is not restored by addition of acid. 

Urobilin. — This has been obtained as a reddish brown 
amorphous substance, but probably not in absolutely 
pure condition. It is slightly soluble in water, readily 
soluble in alcohol and chloroform. The neutral alco- 
hol solutions are characterized by a marked greenish 
fluorescence which is an important means of recogni- 
tion. The acid alcohol solutions are reddish in color, 
the shade varying with the concentration. 

If present in more than minute traces in urine it 
gives characteristic absorption bands in the spectrum 
which have been referred to before. In acid urine the 
center of the dark band is near the Fraunhofer line 
F ; in alkaline urine the center is about midway be- 
tween b and F. 

Urobilin is generally much increased in fevers and 
in some diseases of the liver and heart. Any cause 
tending to break up the red corpuscles, increases uro- 
bilin. It is not always present in sufficient quantity 
in normal urine to be easily recognized. If the quan- 
tity is abnormally large the following test will show it. 

Add ammonia water to strong alkaline reaction and 
filter if necessary. Then add a few drops of solution 
of zinc chloride, but not enough to give a permanent 
precipitate. In this way a zinc salt is formed, which 
shows a peculiar greenish fluorescence. 



COLORING-MATTERS IN URINE 89 

Ammonia generally causes a precipitate of phos- 
phates; hence the direction to filter. If the character- 
istic fluorescence fails to appear the following modifi- 
cation may be tried, which is sufficient to give the 
reaction with most urines. 

Precipitate 200 cc. of urine with basic lead acetate, 
collect the precipitate on a filter, wash it with water 
and dry it. Then wash it with alcohol. 

Finally, digest with alcohol containing a little sul- 
phuric acid, and filter. The filtrate is usually fluores- 
cent. Make it strongly alkaline with ammonia, and 
add solution of zinc chloride. This will give the 
fluorescence referred to above if but little is added, 
while if an excess of the zinc chloride is added, a red- 
dish precipitate falls. 

Urophain. — This is the name given by Heller to a 
substance identical with, or similar to, urobilin. Hel- 
ler gives this test : Take a few cubic centimeters of 
strong sulphuric acid in a conical glass and pour on 
it, drop by drop, about twice as much urine. As the 
two mix, a deep garnet-red is produced. 

This reaction is not, however, characteristic, as sev- 
eral other matters may give it. 

Urohematin is the name given by Harley to a color- 
ing-matter similar to the above. He applies this test : 
Dilute or concentrate the urine so that it is equivalent 
to 1,800 cc. for the twenty-four hours. Take a few 
cubic centimeters in a test-tube or wine-glass, and add 
one-fourth of its volume of strong nitric acid. No 
change of color can be observed if the urohematin is 



90 URINE ANALYSIS 

present in normal amount. If more than this is pres- 
ent various shades from pink to red may be produced. 
The test should be made with cold urine, as with in- 
creased temperature darker colors result. 

Indican and its Reactions. — Although a normal con- 
stituent of urine, indican is found greatly increased 
during the progress of certain diseases and becomes 
therefore a substance of clinical importance. It is 
formed along with other complex compounds in the 
oxidation of indol in presence of sulphuric acid. Indol 
is one of the common products of putrefaction, a 
change brought about in albuminous bodies, usually 
by bacterial agency. Such changes may take place 
in the alimentary canal, and the indol formed becomes 
oxidized to indoxylsulphuric acid or indican, and ap- 
pears as such in the urine. The sulphuric acid neces- 
sary for the production of this body is present in com- 
bination in the system. 

It was formerly supposed that this urinary indican 
is identical with the glucoside indican of the vegeta- 
ble kingdom, from which indigo is obtained. The 
two substances are, however, distinct in composition 
and chemical behavior. The indican of urine, as stated, 
is the sulphuric acid combination of indoxyl, C 8 H 6 . 
N.OH, or the potassium salt of this compound, and 
may be represented by the formula C 8 H 6 NHSO . By 
sublimation or treatment with oxidizing agents this 
yields indigo-blue or indigotin, C i6 H jo N 2 2 . 

If much indican is found it suggests that abnormal 
putrefaction is taking place somewhere in the body. 



COLORING-MATTERS IN URINE. 9 1 

In diseases accompanied by the formation of putrid 
secretions indican usually appears in increased amount, 
and hence the inference derived from its ready detec- 
tion. It is found in increased amount in cancer of 
the stomach or liver, in peritonitis, in some stages of 
pleurisy, in intestinal invagination (whereby the nor- 
mal passage of albuminous and other food products is 
hindered, thus making putrefaction possible) and in 
other diseases. 

Indican is found in normal urines in very small 
amount only. It may, under favorable circumstances, 
be detected as here given : Take about 4 cc. of pure 
hydrochloric acid in a test-tube and add about half as 
much urine, shaking well. A blue or violet color 
shows indican. This test depends on the conversion 
of the indoxyl compound into indigo, but the oxidiz- 
ing action of the acid is not always strong enough to 
bring about the change in presence of other organic 
bodies in the urine. 

A more generally applicable method is this: To 10 
cc. of urine and the same volume of strong pure hy- 
drochloric acid, add 2 or 3 cc. of chloroform. Then 
add, drop by drop, solution of sodium hypochlorite, 
shaking after each addition. The hypochlorite acts 
as an oxidizing agent, liberating the coloring-matter, 
which is then taken up by the chloroform. The oxi- 
dation must not be carried too far ; that is, too much 
hypochlorite must not be added, as it would then de- 
stroy the color as fast as formed. In fact, small traces 
of the product sought might be completely overlooked 
in the process, as the hypochlorite is a very active 



92 URINE ANALYSIS 

oxidizer, the effect going far beyond the production of 
indigo. It has therefore been proposed to use nitric 
acid as the oxidizing- agent. The urine is boiled with 
an equal volume of hydrochloric acid and then a few 
drops of nitric acid are added. The mixture is cooled 
and to it a little chloroform is added and well shaken. 
In presence of indigo the blue color appears. Bro- 
mine water in small amount may be employed in the 
same manner. 

Albumin must be separated by coagulation before 
applying either of these tests, as it develops a blue 
color with hydrochloric acid. The amount of indican 
normally present in urine is said to vary between 5 
and 20 milligrams daily. The chloroform layer in the 
bottom of the test-tube in the above test shows roughly 
by the depth of color developed the amount of indican 
present. It is necessary to use good hypochlorite for 
that test as with a weak solution the oxidation may 
fail to take place. 

Abnormal Coloring=matters 

In disease several other coloring-matters may ap- 
pear in urine, the most important of which are those 
of the bile and blood. 

As abnormal colors must be classed, also, many prod- 
ucts taken into the stomach with the food or as reme- 
dies and which appear directly in the urine or give 
rise to marked coloration on the addition of reagents. 

Biliary Coloring-matters 

These are found in the urine in jaundice and may 
be traced to the stoppage of the bile ducts of the liver 



COLORING-MATTERS IN TRINE 93 

as in common jaundice and to other causes having no 
connection with a disorder of the liver. The appear- 
ance of these coloring-matters in the urine is there- 
fore a symptom of different diseases, although perhaps 
most commonly associated with an abnormality in the 
flow of the bile. Jaundice may sometimes be traced 
to a disintegration of the red corpuscles in the blood 
and consequent liberation of derived coloring-matters. 
Biliary urine has generally a characteristic greenish 
yellow color sometimes tinged with brown. The froth 
from such urine is readily recognized by its yellow 
color, which is often a sufficient test in itself. Among 
the chemical tests the following are the best known. 

Gmelin's Test. — This is easily performed and de- 
pends on the oxidation of bilirubin, the pigment 
commonly present in fresh jaundice urine, by nitrous 
acid. Pour in a test-tube about 5 cc. of the urine un- 
der examination and by means of a pipette introduce 
below it an equal volume of strong nitric acid mixed 
with nitrous. This should be carefully done so as to 
avoid mixing the liquids much. At the junction of 
the two liquids, if bile is present, several colored rings 
appear of which the green due to biliverdin is most 
characteristic. The bands or rings appear above the 
acid in this order, yellowish red, red, violet, blue, and 
green. The last is essential. It must be remembered 
that nitric acid gives the other colors at times with 
urine free from bile, but green is characteristic of the 
latter. 

Fleischl modified this test by mixing the urine with 
a strong solution of sodium nitrate and then adding 



94 URINE ANALYSIS 

strong sulphuric acid carefully. This settles below 
the urine and decomposes the nitrate at the point of 
contact liberating the necessary nitric and nitrous 
acids for the oxidation as before. This method is a 
very good one. 

In another modification, urine is dropped on a plas- 
ter of Paris disk and then a few drops of the oxidizing 
mixture of nitric and nitrous acids is placed in its 
center. The same play of colors appears as before. 

Trousseau's Test. — Add to some urine in a test-tube 
a few drops of tincture of iodine, allowing the iodine 
to float on the urine. If bile pigments are present a 
green color is produced when the iodine touches the 
urine, and persists some hours. Care must be taken 
to avoid using an excess of the iodine if the liquids are 
allowed to mix. In this case with the proper amount 
of the tincture the whole urine appears green. 

Heller's Test. — Take 5 or 6 cc. of pure strong hydro- 
chloric acid in a conical glass and add enough of the 
urine to give it a faint color on mixing. Now add 
pure nitric acid by means of a pipette so as to bring 
the latter under the mixture of hydrochloric acid and 
urine. The colored rings appear as in the Gmelin 
test and on shaking can be followed through the 
liquid. 

The Detection of Traces. — To 100 cc. of the urine 
add 10 cc. of pure chloroform and shake gently until 
the latter is colored. By means of a pipette withdraw 
a small part of the chloroform and mix it in a test- 
tube with 10 cc. of strong pure hydrochloric acid. Add 



COLORING-MATTERS IN URINE. 95 

nitric acid as in the other tests and shake. With bile 
present, the oxidation colors appear slowly in the 
chloroform, the green being the deciding tint. 

The Diazo Reaction. — Ehrlich and others have called 
attention to the behavior of many nrines with solution 
of diazobenzene sulphonic acid which often has im- 
portance in diagnosis. Normal urine treated with a 
weak solution of this reagent shows no marked change, 
but in several pathological conditions after adding the 
acid and saturating with ammonia a deep carmine- or 
scarlet-red color appears, followed by greenish or vio- 
let precipitation. 

This reaction depends on the combination of the 
sulphonic acid of diazobenzene with some aromatic 
compound found in the urine in pathological condi- 
tion. At one time it was supposed to have special 
significance in the diagnosis of typhoid fever, but it 
now appears that in many diseases of the intestinal 
tract the urine receives traces of complex aromatic 
products of bacterial origin which respond to the test. 
It has, therefore, general, rather than special, signifi- 
cance. 

As the diazobenzene sulphonic acid is not very 
stable, it is not convenient to use, and a reagent is 
made which, in its application, is its chemical equiva- 
lent. The reagent is prepared by dissolving i gram 
of sulphanilic acid in 200 cc. of water with the addi- 
tion of 10 cc. of pure hydrochloric acid. Another so- 
lution is made by dissolving 1 gram of sodium nitrite 
in 200 cc. of water. To make the test take 50 cc. of 
the first solution, add 5 cc. of the nitrite solution 



96 URINE ANALYSIS 

and then 50 cc. of urine. Ammonia is then added in 
sufficient quantity to impart a strong alkaline reaction 
after thorough shaking. A scarlet- red color is the 
result if the urine in question contains the abnormal 
products referred to. 

Melanin. — In the urine of patients suffering from 
melanotic cancer a dark color sometimes appears which 
may become almost black on exposure to air. Treat- 
ment of the urine with oxidizing reagents increases 
the effect ; with bromine water a yellowish color is 
first given followed by black. The coloring substance 
may be even thrown out as a brownish black sediment. 
In urine containing this body, to which the name 
melanin has been given, yellowish brown precipitates 
are produced by the addition of baryta water and basic 
lead acetate, but the coloring-matter itself has never 
been isolated in pure condition. Its recognition may 
have diagnostic value in cases where the melanotic 
condition may not otherwise be suspected. 

Blood Colorixg-matters 

As these appear in the urine they may be derived 
from different sources. We may have, first, color due 
to the presence of blood corpuscles themselves some- 
times in nearly fresh condition. There may be enough 
blood present to impart to the urine a marked red color 
and it may be derived from the kidney, bladder, ure- 
thra, or other part of the urinary tract. In blood from 
a fresh lesion the corpuscles usually appear in clearer 
outline than is the case when they have remained long 
in contact with the urine. 



COLORING-MATTERS IN URINE 97 

The presence of blood may be detected by several 
methods. The corpuscles are often easily recognized 
by the microscope in the sediment deposited when the 
urine is allowed to stand, as will be explained in a 
following chapter. Then we can make use of the 
spectroscope, by which means the characteristic absorp- 
tion bands of oxyhemoglobin are detected, as explained 
in works on chemical physiology. If urine contain- 
ing blood is treated with a few drops of ammonium 
sulphide and very gently warmed, the spectrum of re- 
duced hemoglobin is given. 

Sometimes the coloring-matters alone without the 
corpuscles can be found. This is the case when the 
latter become disintegrated, the more stable and solu- 
ble hemoglobin passing into solution while the stroma 
disappears by decomposition. The condition in which 
blood itself is present, and can be recognized by the 
microscope, is known as hematuria, while the condi- 
tion characterized by the presence of the coloring-sub- 
stance only is called hemoglobinuria. Urine contain- 
ing the products of blood decomposition is often 
brown or smoky colored. 

In urine, hemoglobin frequently undergoes two de- 
compositions. It may become converted into methemo- 
globin, or it may suffer a complete modification, break- 
ing up into hematin and a body resembling globulin. 
Hematin is best recognized by spectroscopic examina- 
tion, as it gives a spectrum different from hemoglobin. 
This modified product is said to occur in urine in cases 
of poisoning by hydrogen arsenide. 
7 



98 URINE ANALYSIS 

The following are the best chemical tests for the 
recognition of these bodies : 

Heller's Test. — Treat the urine with solution of so- 
dium or potassium hydroxide, and heat to boiling. 
This produces a precipitate of the earth}' phosphates 
which in subsiding carry down coloring-matters. If 
a precipitate does not separate readily it may be 
hastened by adding two or three drops of magnesia 
mixture. Hemoglobin, when present, is decomposed 
by this treatment with separation of hematin, which 
in turn settles down with the phosphates, imparting 
a red color to the precipitate. 

Struve's Test. — Make the urine slightly alkaline 
with sodium hydroxide solution, and then add enough 
solution of tannic acid in acetic acid to change the re- 
action. If hemoglobin is present a dark brown pre- 
cipitate of hematin tannate settles out. The test is a 
good one, and easily performed, but is not sufficiently 
delicate for the detection of small traces of hemoglo- 
bin directly. By collecting the precipitate on a filter 
and washing it, it may be used for two confirmatory 
tests. One of these depends on the formation of he- 
min crystals and is made in this manner : Place a small 
portion of the precipitate on a microscopic glass slide 
and add a minute crystal of sodium chloride. Then 
add a large drop of glacial acetic acid and cover with 
a cover glass. Warm very gently over a small flame. 
When the acid, salt, and precipitate have become thor- 
oughly mixed allow the slide to cool. Small rhombic 



COLORING-MATTERS IN URINE 99 

crystals-of hemin should now appear, which are best 
seen under a microscope. 

The washed precipitate may also be ashed and used 
for an iron test. The ash should be dissolved in a 
little pure hydrochloric acid in a porcelain dish and 
tested by the addition of potassium ferrocyanide and 
ferricyanide to give the well-known reaction. This 
test presupposes purity and freedom from traces of iron 
in the reagents used. 

Almen's Guaiacum Test. — In a test-tube mix equal 
volumes of fresh tincture of guaiacum and ozonized 
turpentine, — 2 or 3 cc. of each will suffice. The mix- 
ture, if made of proper materials, must not show a 
green or blue color after thoroughly shaking. Now 
add a few cubic centimeters of the urine to be tested, 
a drop at a time, and agitate after each addition. If 
hemoglobin is present it causes the oxidizing material 
of the ozonized turpentine (probably hydrogen per- 
oxide) to act on the precipitated guaiacum resin, im- 
parting to it first a greenish, and finally a blue color. 
Old and alkaline urine must be made faintly acid before 
performing the test. Pus in the urine gives a some- 
what similar reaction, and a few other bodies, very 
seldom present, interfere. The test is very delicate, 
and if it gives a negative result it is safe to conclude 
that blood is absent. 

Vegetable and Other Colors 

It has long been known that many peculiar coloring- 
matters enter the urine from substances taken as reme- 



IOO URINE ANALYSIS 

dies and sometimes as food. A few of the more com- 
mon of these colors will be mentioned here. 

Chrysophanic Acid. — This compleN organic acid is 
found in the root of several kinds of rhubarb, in senna 
leaves, in certain lichens, and elsewhere. After the 
administration of any of these substances the nrine 
becomes more highly colored, being a brighter yellow r 
if acid and yellowish red when made alkaline. When 
phosphates are precipitated by addition of alkali they 
appear red in presence of chrysophanic acid, as they 
do with blood. But the latter can be easily distin- 
guished by the other tests already given. Urine con- 
taining this acid is further distinguished by giving a 
red precipitate with solution of lead acetate. 

Santonin. — This crystalline principle is found in the 
nnexpanded flowers of Levant wormseed, and when 
administered as a remedy produces a characteristic 
change in the color of the nrine. The color becomes 
a deep yellow which tnrns red with alkalies, as in the 
case of chrysophanic acid. If the colored alkaline 
urine is shaken with amyl alcohol the coloring-matter 
from the santonin leaves the urine and passes into the 
alcohol, but the color from chrysophanic acid is only 
very slightly soluble in amyl alcohol and remains with 
the urine when the same treatment is applied. 

Salicylic Acid. — The urine of persons taking this 
substance has usually a grayish smoky tinge which 
becomes blue on addition of solution of ferric chloride 
if more than traces are present. 



COLORING-MATTERS IN URINE IOI 

Salicylic acid is excreted in the free state or as a 
salicylate of sodium or potassium mainly; a small 
portion seems to pass into other compounds. But as 
the iron reaction is very delicate minute amounts of 
the free or combined acid can be found. Enough fer- 
ric chloride must be added to be in excess of what 
would combine with the phosphates present, otherwise 
a sharp reaction may not be secured. 

Phenols. — Several phenol bodies as carbolic acid, 
hydroquinol, resorcinol, pyrocatechol and others some- 
times find their way into the urine, to which they im- 
part a dark color on standing exposed to the air. This 
change of color is said to be due to the formation of 
oxidation products of hydroquinol. From urine dark. 
ened in this manner phenols have been recovered by 
making acid with sulphuric acid and then distilling 
with steam. 

Some of these phenols, in traces, are undoubtedly 
normal urinary constituents, but pathologically they 
may appear in increased amount, and also after admin- 
istration of various aromatic remedies. The external 
application of common phenol is followed by the ap- 
pearance of traces in the urine, which is disclosed usu- 
ally only after the latter has stood some time exposed 
to the air. Many reactions are given in the books for 
the recognition of traces of phenol, but as a rule they 
cannot be applied directly to urine. For detection 
here a liter may be distilled after the addition of 25 
cc. of strong sulphuric acid. To a portion of the dis- 
tillate add enough bromine water to impart a yellow- 



102 URINE ANALYSIS 

ish color. In presence of phenol a light yellowish 
precipitate of tribromphenol appears. To a second 
portion of the distillate add a few drops of ferric chlo- 
ride solution; with phenol this gives a purple color. 
The same reaction is given by other phenols and by 
salicylic acid. To separate the latter a portion of the 
distillate is made alkaline with sodium carbonate and 
the solution so obtained is shaken with ether, the 
operation being repeated several times. The salicylic 
acid is held, while phenols pass into the ethereal solu- 
tion. After evaporating the ether and taking up with 
water the tests for phenols may be made. 

Other Colors. — Blueberries, carrots, and several other 
common vegetable foods give deep color to the urine. 
It is not always possible to recognize the coloring-sub- 
stances in these cases. Such urine usually becomes 
yellow 7 with acids and reddish with alkalies. It is oc- 
casionally possible to identify the color by means of 
the spectroscope, as the absorption spectra of some of 
these products have been studied. 

The Detection of the Bile Acids 

It sometimes happens that the physician desires in- 
formation regarding the presence of the biliary acids 
as well as the pigments in the urine. This informa- 
tion, however, is not easily secured because there is no 
simple test which can be applied directly to the urine 
which will give a certain indication of the presence of 
these acids. They must first be separated from the 
large amount of other substances present, which can 
be done in this way (Neukomm) : 



COLORING-MATTERS IN URINE 103 

Evaporate 300 to 500 cc. of urine nearly to dryness ; 
extract with ordinary alcohol, evaporate this solution, 
and extract the residue with absolute alcohol. 

Evaporate this and take up the new residue with 
water. Precipitate the solution by lead acetate, 
avoiding excess ; allow to settle, wash the precipitate 
with water on a filter, and dry in folds of bibulous 
paper. This leaves an impure lead salt of the acids. 
Extract it with hot alcohol, and filter; add sodium 
carbonate to the filtrate, evaporate to dryness and 
extract the sodium salt, thus formed, with absolute 
alcohol. Evaporate again, add some water and apply 
the Pettenkofer test, as follows : 

To the solution add one or two drops of a 20 per 
cent, cane-sugar solution, and then some strong sul- 
phuric acid, slowly to avoid heating. 

It is best to immerse the test-tube in water to keep 
the temperature below 60 ° C. As the acid mixes 
with the liquid a violet or purple color is produced. 
It has been shown that this, like the a-naphthol test 
for dextrose is a furfural reaction, the furfural formed 
from the mixed sugar and acid combining with the 
acids of the bile. It has even been proposed to use a 
dilute solution (one-tenth per cent.) of furfural instead 
of the sugar in the test. 

Kuelz recommends to evaporate the solution on a 
water-bath to dryness, to moisten the residue with a 
drop of dilute sugar solution, and then with a drop of 
the strong acid. The color appears almost immedi- 
ately, but can be sharpened by heating the evapora- 
ting dish a few seconds on the water-bath. 



104 URINE ANALYSIS 

Applying either of these tests directly to urine is 
unsafe, as the coloring, and other, matters present would 
intrefere very much with the reaction. 



Chapter V 

DETERMINATION OF URIC ACID. HIPPURIC ACID 

Uric acid, CHNO, occurs normally in urine com- 

' 5 4 4 3' f 

bined with sodium, potassium, magnesium, or ammo- 
nium. The absolute amount excreted daily is small 
but quite variable, depending on many conditions not 
well understood. In health the amount passed daily 
seems to vary between 0.2 gram and 1 gram. These 
limits may not be correct, however, as many of the 
older determinations were made by inaccurate methods. 

Regarding the clinical significance of variations in 
the amounts of uric acid passed, our knowledge is still 
very defective. It is generally held that there is a 
considerable increase in the excreted uric acid in fevers 
and in diseases characterized by diminished respira- 
tion and consequently imperfect oxidation. In leuce- 
mia there is a pronounced and characteristic increase 
of uric acid. Certain writers have attempted to con- 
nect a decreased elimination of uric acid with an ac- 
cumulation of the same in the blood, giving rise to 
numerous disorders of which gout may be mentioned 
as one in which the connection has been, apparently, 
clearly shown. Great variations in the excreted uric 
acid seem to be characteristic of a train of disorders, 
rather than of a single one. 

From recent investigations it appears that the ratio 
of excreted urea to uric acid is in health not far from 



106 URINE ANALYSIS 

35 : i, and that variations in this ratio are of greater 
moment than are variations in the absolute amount 
of the acid. Both must be considered as normal end 
products of nitrogenous metabolism, contrary to the 
older view that uric acid is the antecedent of urea, and 
that the amount of the former found in the urine rep- 
resents merely that which failed to be completely oxi- 
dized. A marked change in the above ratio, 35 : 1, 
by increase of the uric acid is characteristic of a con- 
dition which is somewhat indefinitely called the uric 
acid diathesis. 

In the recognition of uric acid the following points 
may be noted : When present in large amount it fre- 
quently precipitates from the urine in the free form, 
or as acid urates which have a yellowish color. When 
the amount present is small it may be found by acidi- 
fying with hydrochloric acid and then allowing the 
urine to stand some hours in a cool place ; uric acid 
crystals separate. In mixed sediments it may be rec- 
ognized by this test : 

Murexid Test. — Throw the sediment on a filter and 
wash once with water. Place the residue in a porce- 
lain dish, add a drop of strong nitric acid, and evapo- 
rate to dryness on the water-bath. A yellow or brown 
mass is obtained, and this touched with a drop of am- 
monia water turns purple. 

Unless the uric acid or urate is present in the sedi- 
ment in fine granular form its recognition by the mi- 
croscope is very simple. Illustrations of the forms of 
uric acid and certain urates are given in the chapter 
on the sediments. 



DETERMINATION OF URIC ACID 107 

The Amount of Uric Acid 

For the determination of the amount of the acid in 
the urine we have the choice of several methods, not 
one of which is very convenient or of the greatest ac- 
curacy. The first of these depends on the fact referred 
to above, that hydrochloric acid liberates uric acid 
from its combination, precipitating it in crystalline 
form. 

Precipitation Test. — Measure out 200 cc. of urine 
and add to it 20 cc. of strong hydrochloric acid. Mix 
thoroughly and set aside in a cool place for about forty- 
eight hours. At the end of this time collect the red- 
dish-yellow deposit on a weighed filter, wash it with 
a little cold water, dry, and weigh. Xot over 30 or 40 
cc. of water should be used in the washing. The pre- 
cipitated uric acid is not pure, holding coloring and 
other substances which increase its weight. On the 
other hand, it is soluble to some extent even in cold 
acidulated water so that not the whole of it is ob- 
tained on the filter and a correction must be made. It 
is usually recommended to add to the weight obtained 
4.8 mg. for each 100 cc. of filtrate and washings. 

If the urine under examination contains albumin, 
the latter must be coagulated by heating with a drop 
or two of acetic acid and filtered out, before the test 
is made. If the urine is very cold to begin with and 
has a sediment of urates, the latter must be brought 
into solution by warming before beginning the test. 
To prevent precipitation of phosphates during the 



108 URINE ANALYSIS 

warming a few drops of hydrochloric acid may be 
added. This method is at best only a rough approxi- 
mation, but is the one by which most of our results 
have been obtained. The following gives better re- 
sults : 

Salkowski-Ludwig Method. — The determination here 
is based on the fact that uric acid gives a very insolu- 
ble precipitate with ammoniacal solution of silver ni- 
trate, from which precipitate, after filtration and wash- 
ing, the acid may be readily separated, brought into 
concentrated solution, reprecipitated and weighed. 

In using the method the following solutions are 
required : 

( a) Ammoniacal Silver Nitrate.— Dissolve 25 grams of 
silver nitrate in 100 cc. of distilled water, add am- 
monia water until the precipitate which appears 
at first is completely redissolved, leaving a clear 
solution. Make this up to 1000 cc. with distilled 
water and keep in a dark bottle or away from the 
light. 

(b) Magnesia Mixture. — Made as described in the ap- 
pendix. It must be strongly alkaline and clear, 
or nearly so. 

(V) Solution of Potassium or Sodium Sulphide. — The pure 
crystals of sodium sulphide obtained from dealers 
in chemicals may be used by dissolving 25 to 30 
grams (Na 2 S.9H 2 0) in 1000 cc. of distilled water. 
A solution may be made, also, by dissolving 10 
grams of pure sodium h3'droxide in 1000 cc. of 
water, and converting this into sulphide, which is 






DETERMINATION OF URIC ACID I09 

done as follows : Divide the solution into two equal 
portions. Saturate one thoroughly with r^dro- 
gen sulphide and to this then add the other half. 
Keep in a glass-stoppered bottle, the stopper par- 
affined. 

To make the test, measure out 200 cc. of the urine 
and transfer to a beaker. Add 20 cc. of the silver so- 
lution, («), to an equal volume of the magnesia mix- 
ture, ($), and then ammonia enough is added to clear 
up any precipitate which forms. This clear mixture 
is now poured into the urine in the beaker and the 
whole well stirred. A precipitate of silver urate forms 
along with silver and earthy phosphates. The excess 
of ammonia prevents the precipitation of silver chlo- 
ride. Silver urate is quite insoluble in ammonia ; it 
is gelatinous alone and does not settle very well but 
the phosphate precipitate corrects this difficulty to 
some extent. The beaker is allowed to stand at rest 
about an hour, after which the contents are filtered 
and the precipitate washed with weak ammonia on 
the filter. To do this the ammonia is sprayed into 
the beaker from a wash-bottle and rinsed around thor- 
oughly. This is done several times, the liquid being 
poured on the filter. Where available a Gooch cruci- 
ble serves admirably for the collection of the precipi- 
tate as the filtration is slow on paper without aspira- 
tion. It is not necessary to remove any of the precipi- 
tate which clings to the beaker, as will be seen. When 
the washing is complete transfer the precipitate and 
filter-paper, or asbestos if the Gooch crucible is used, 
back to the beaker and pour over it a boiling mixture 



IIO URINE ANALYSIS 

of 20 cc. of the sulphide solution, (<r), and 20 ec. of 
distilled water. Stir up thoroughly, allow to stand 
some time and then add 50 ce. of boiling water. Place 
the beaker on a sand-bath or gauze and bring the con- 
tents to boiling, stirring continually. Keep hot some 
minutes and then allow to stand until cold, the pre- 
cipitate being stirred meanwhile occasionally. 

The treatment with the sulphide solution decom- 
poses the silver urate with precipitation of black in- 
soluble silver sulphide, the uric acid remaining in so- 
lution as soluble urate. The cooled liquid is filtered 
into a porcelain dish, and the precipitate washed with 
warm water, the washings going also into the dish. 
Enough hydrochloric acid is now added to combine 
with all the bases present and liberate the uric acid, 
which is the case when the liquid becomes acid in re- 
action. It is now slowly evaporated to a volume of 
about 10 cc, best on a water-bath, and then allowed 
to stand an hour for the complete separation of the 
uric acid. This is then collected on a weighed Gooch 
crucible, the crystals being transferred gradually by 
aid of the filtered liquid. When the crystals are on 
the asbestos they are washed with a little acidulated 
water several times. The crucible is then dried at 
ioo°, put back in the funnel and treated with a small 
amount of pure carbon disulphide to remove traces of 
sulphur separated on decomposing the alkali sulphide. 
Finally, wash with ether, dry at ioo° C. and weigh. 
The results are always a little low, but fairly regular. 

As the acid is finally precipitated from a very small 
volume of liquid and but little water is used in wash- 



DETERMINATION OF URIC ACID III 

ing, no correction need be made for solubility, as in 
the first process described. While simple enough in 
principle and easily carried ont considerable time is 
required for the performance of all the operations in- 
volved in the method. 

The following method is free from this objection 
and is equally accurate : 

Haycraft Method. — This depends on the precipita- 
tion of the uric acid, as silver urate, and its subsequent 
titration by standard solution of ammonium thiocya- 
nate. The following solutions are required : 

(a) Ammoniacal Solution of Silver Nitrate. — Dissolve 5 
grams of crystals in 100 cc. of water and then add 
enough ammonia water to give the solution a strong 
alkaline reaction. Make up to 200 cc. with the 
ammonia. 

(b) Ammonium Thiocyanate. — This solution is made as 
described later for the determination of chlorides 
in urine by the Volhard method. It is given just 
one-fifth the strength there described and may be 
made by diluting 100 cc. of that solution to 500 
cc. in a measuring flask. • 

(V) Ammonium Ferric Sulphate (ferric alum). — Satu- 
rated solution as indicator. Described under the 
chlorine test. 

It has been shown by Dr. Haycraft that silver com- 
bines with uric acid in constant and definite propor- 
tion; viz., one atom of silver to one molecule of the 
acid, or 107.9 P ar ts of silver to 168.2 of the acid, giv- 
ing the formula AgC H N O . 

& & 5 3 4 3 



112 URINE ANALYSIS 

This precipitate dissolves in dilute nitric acid and 
if the solution so obtained is treated with the ammo- 
nium thiocyanate the following reaction takes place : 

AgC 5 H 3 N 4 3 + NH 4 SCN == 

AgSCN + NH 4 C 5 H 3 N 4 3 - 

From this it follows that i cc. of a fiftieth normal, 
(^\), solution of the thiocyanate liberates and indicates 
0.00336 gram of uric acid. 

It is fully explained under the chlorine test that if a 
solution of a thiocyanate is added to a solution of a silver 
salt containing nitric acid and ferric sulphate, a complete 
reaction takes place between the thiocyanate and silver 
before the characteristic reaction between the former 
salt and the ferric compound appears. In other words, 
the thiocyanate and the silver combine first and then 
any further amount of thiocyanate added unites with 
the iron, producing a red color (of ferric thiocyanate) 
indicating the completion of the first reaction. 

With these general explanations the process will now 
be understood. 

Measure out 50 cc. of the urine and warm it gently 
if it contains a sediment of urates. Add 3 to 4 grams 
of pure sodium bicarbonate and then ammonia enough 
to give a strong alkaline reaction. This may give a 
precipitate of phosphates which need not be heeded. 
Next add 5 cc. of the silver solution, (#), and mix 
thoroughly. This produces a precipitate of silver 
urate along with the bulky phosphates thrown down 
by the ammonia. Allow to stand half an hour and 
then filter. A paper filter and funnel may be used in 



DETERMINATION OF URIC ACID II3 

the usual manner, but much better results are obtained 
by the use of the Gooch crucible and asbestos with aid 
of an aspirator. Rinse the sides of the beaker thor- 
oughly with weak ammonia and pour this on the pre- 
cipitate in the funnel or crucible. Continue the wash- 
ing of the precipitate with weak ammonia water until 
all traces of silver are washed out, as may be shown 
by allowing a few drops of the filtering washings to 
fall into some dilute hydrochloric acid in a test-tube. 
The washing is complete when a cloudiness is no 
longer obtained in this test. 

Now pour some pure dilute nitric acid into the 
beaker in which the precipitation was made, and 
which was washed free from silver by the ammonia, 
and shake it around until any traces of the silver urate 
precipitate are dissolved. Put the funnel or Gooch 
crucible over a clean receptacle and pour this acid 
liquid on the precipitate. Silver urate dissolves com- 
pletely in dilute nitric acid, and enough of this is 
added, a little at a time, to bring about complete solu- 
tion. It now remains to titrate the silver in this so- 
lution. To this end add 5 cc. of the ferric alum solu- 
tion, and if the mixture is not clear and colorless, 
about 2 cc. of pure strong nitric acid. Then from a 
burette run in the thiocyanate, (b), a little at a time, 
shaking after each addition until a faint red shade 
of ferric thiocyanate becomes permanent. Toward 
the end of the titration a red appears as each drop of 
liquid from the burette falls into the silver solution 
below, but this color fades out on shaking and does 
not persist until the last particle of silver has been 
8 



114 URINE ANALYSIS 

taken up by the thiocyanate. Supposing now that 
15 ce. of the latter solution are required to reach this 
point we have 15 X 0.00336 = 0.0504 gram as the 
amount of uric acid in the 50 cc. of urine taken. A 
volume as large as this would seldom be required; 5 to 
10 cc, corresponding to 16.8 to 33.6 milligrams, is 
usually sufficient. 

The method gives results which are a little too high 
as the silver carries down traces of other bodies as 
well as uric acid ; but the error is not great enough 
to interfere with the practical application of the pro- 
cess where the best obtainable results are desired. The 
washing of the precipitate of silver urate is the point 
which requires the greatest care. A little practice 
will show how this can be best done. 

Fokker-Hopkins Method. — It has long been known 
that uric acid may be very completely precipitated 
from urine in the form of acid ammonium urate by 
the addition of an ammonium salt. The method of 
precipitation as first described by Fokker was not sat- 
isfactory, and was modified by Salkowski. More re- 
cently it has been improved by Hopkins, and in this 
form may be carried out as follows : Add to 100 cc. of 
urine enough pure, finely granular or powdered am- 
monium chloride to completely saturate it. About 30 
grams of the salt will be necessary for this. The urine 
must be well stirred as the chloride is added to facili- 
tate its solution. When no more will dissolve the so- 
lution is allowed to stand about two hours and should 
meanwhile be stirred occasionally. The precipitate 



DETERMINATION OF URIC ACID 115 

which settles is collected on a small filter and well 
washed with a saturated solution of ammonium sul- 
phate. In this washing the precipitate is freed from 
some coloring, and other, matters and is then ready for 
further treatment. Two methods are available for 
rapid work. In the first the filter with the precipi- 
tate is placed over a beaker or flask, and through a 
hole made in the bottom of the paper with a glass rod 
the precipitate is washed down into the receptacle. 
This can be easily done with the aid of a fine jet 
from a wash-bottle. Use about ioo cc. of water and 
to the turbid liquid add now 20 cc. of pure colorless 
sulphuric acid. This produces a high temperature 
and dissolves the uric acid. The amount of the latter 
is now found by titration with twentieth normal per- 
manganate solution (1.58 1 grains of KMnO per liter) 
as follows : The permanganate solution is contained 
in a burette and without delay is run into the hot uric 
acid solution. A reduction of the reagent, with loss 
of color, follows. When the uric acid is fully oxidized 
a farther addition of permanganate leaves a pink tinge 
in the liquid. The addition of the standard reagent 
from the burette should cease as soon as a pink tinge 
is reached, which is permanent two seconds after good 
shaking. By waiting a longer interval the color fades 
and more solution must be added from the burette. If 
the reaction is stopped with the first decided tinge ob- 
tained, as explained, for each cubic centimeter of per- 
manganate used from the burette, 3.75 milligrams of 
uric acid may be calculated as present. In illustra- 
tion, suppose we start with 100 cc. of urine and pre- 



Il6 URINE ANALYSIS 

cipitate, wash and dissolve as described. If now we 
run 12.5 cc. of the twentieth normal permanganate 
solution into the hot uric acid solution to obtain the 
pink color, the amount of this acid present is 12.5 X 
3.75 = 46.87 milligrams in the 100 cc. Urine con- 
tains traces of other bodies which are precipitated with 
the uric acid, but in amount so small that their effect 
may be practically neglected. 

Instead of oxidizing the uric acid precipitate with 
permanganate solution it may be titrated by means of 
weak standard alkali solution, preferably twentieth 
normal. When this is to be done it is best to start 
with 200 cc. of urine and precipitate and wash as be- 
fore. The precipitate is boiled up with 30 cc. of 
twentieth-normal hydrochloric or sulphuric acid and 
enough water to make about 200 cc. Then two drops 
of a weak methyl orange solution are added and finally, 
from a burette, the twentieth-normal alkali until the 
reddish pink color changes to orange-yellow. Part 
of the acid added is used in decomposing the acid 
ammonium urate, and is therefore a measure of the 
latter. The alkali run in measures the excess of 
hydrochloric acid, as uric acid is neutral toward 
methyl orange. Therefore, subtract from the 30 cc. 
of acid the volume of alkali required to give the final 
reaction ; the remainder measures the alkali actually 
required for the uric acid. For each cubic centimeter 
of alkali calculate 8.4 milligrams of uric acid. In 
illustration, suppose we precipitate 200 cc. of urine, 
collect and wash the precipitate and dissolve as 
described, adding 30 cc. of twentieth-normal acid. 



DETERMINATION OF URIC ACID 117 

If now we run in 13.5 cc. of twentieth-normal alkali 
we have 30 — 13.5 = 16.5 cc. of alkali actually re- 
quired for the uric acid. Then, 16.5 X 8.4 = 138.6 
milligrams in 200 cc. 

In a following chapter something will be said about 
the occurrence of uric acid sediments in urine. Under 
certain conditions a large part of this acid present may 
separate in the free form or in combination as slightly 
soluble urate, readily recognizable by the microscope. 

Hippuric Acid 

This acid, which is benzoyl-amidoacetic acid, 
C H NO , is found in very small amount normally in 
human urine, and is the chief nitrogenous product in 
the urine of the herbivora. It is increased in human 
urine by a diet of aromatic vegetable substances, but 
is seldom abundant enough to have clinical impor- 
tance. The amount excreted varies usually between 
0.1 gram and 1 gram daily. Even larger amounts 
have been reported, but the excretion seems to be con- 
nected with the consumption of unusual fruits or vege- 
tables. . The effect of cranberries is especially charac- 
teristic here. 

It is also known that benzoic acid and benzoates 
taken internally become changed in the body to hip- 
puric acid and are so excreted. Under such circum- 
stances the percentage amount may become relatively 
very large, even as much as 1.5 to 2 per cent. 

For the detection of hippuric acid in urine it is best 
to take 1000 to 1500 cc. and make slightly alkaline, if 
acid, with sodium carbonate. Filter and evaporate 



Il8 URINE ANALYSIS 

the filtrate nearly to dryness. Extract the residue 
several times with small portions of alcohol, evaporate 
the alcohol and treat the aqueous solution left with 
enough hydrochloric acid to impart a sharp acid re- 
action. Pour this solution into a separatory funnel 
and shake out with acetic ether five or six times. 
This dissolves the hippuric acid. The different por- 
tions of acetic ether are united and washed in the fun- 
nel with water. The so-purified ethereal solution is 
evaporated slowly to deposit the hippuric acid. If 
traces of fat appear to be present wash this residue 
with petroleum ether. Then dissolve the remaining 
acid in hot water, filter and allow the solution to 
evaporate spontaneously, or at a temperature not above 
50 , to crystallization. Hippuric acid may be recog- 
nized by the microscope, as shown later, or by this 
chemical test. To some of the crystalline product in 
a dish add a little strong nitric acid and evaporate to 
dryness. By heating now carefully to a higher tem- 
perature a strong odor of nitrobenzene is developed. 
In this reaction nitrobenzoic acid is produced from the 
hippuric acid and at a higher temperature yields carbon 
dioxide and nitrobenzene. 



Chapter VI 

UREA 

Urea, C0(NH 2 ) 2 , is the most important nitrogenous 
substance excreted in human urine. A large part of 
the nitrogen of our food is normally converted into 
urea for elimination from the body, but how this con- 
version takes place, or where, is not definitely known. 
It has been shown that in the liver it may be produced 
from ammonium carbonate and certain other compara- 
tively simple bodies, but the connection with the an- 
tecedent and much more complex proteid bodies is 
quite obscure. Experiments which have been carried 
out by physiologists to determine the part played by 
the kidneys in the elimination of urea have led to 
very contradictory results. It has been found that 
the synthesis of hippuric acid from benzoic acid and 
glycocoll may be effected in the extirpated kidney, 
but attempts to form urea in an analogous manner 
from blood charged with ammonium carbonate led 
only to negative results, from which it would appear 
that the function of the kidneys, as far as the elimi- 
nation of urea is concerned, is merely a mechanical 
one. Other investigations, however, have led certain 
observers to the view that the cells of the kidney are 
active in the formation of urea from products coming 
from the disintegration of elements of the blood, if 



120 URINE ANALYSIS 

not from ammonium salts. After extirpation of the 
kidney there is observed an accumulation of urea in 
the blood of the living subject, which is a point not 
without importance, as it shows that this organ can 
not be the only seat of the reaction of urea production. 

Not far from 85 per cent, of the nitrogen consumed 
as food is excreted as urea, but the absolute amount 
of the latter passed in a day is exceedingly variable. 
In the urine of the average man it is between 30 and 
40 grams while in the urine of women it is less. The 
variations depend mainly on the diet, the urea being 
highest with a diet rich in meat, eggs, beans, peas, 
and similar vegetables, and low with a diet of fruits, 
bread, and potatoes. The percentage amount of urea 
depends further on the volume of the urine passed in 
a day and may vary from a change in the amount of 
water consumed and also from different losses by per- 
spiration. The percentage amount of urea depends 
also on the time when the urine is voided. A deter- 
mination of value should therefore be made on the 
mixed urine of the twenty-four hours. 

It is usually assumed that 2 per cent, is the average 
amount excreted in health, but this is probably low. 
While the variations from this mean are great in 
health, they are much more marked in pathological 
conditions. 

Urea is increased in total amount, although it may 
be diminished in percentage in diabetes mellitus and 
insipidus and also in fevers. It has been found to be 
increased in cases of poisoning by heavy metals, but 
why is not clearly demonstrated. 



UREA I 2 I 

Clinically, the increase in diabetes and fevers is of 
the greatest interest because we have here evidence of 
increased consumption of the nitrogenous tissues of 
the body. A diminished elimination of urea has been 
observed in acute yellow atrophy of the liver and in 
other diseases of that organ. This has been taken to 
indicate that the liver may be the place of formation 
of urea. In cases of malnutrition in general the abso- 
lute and percentage amount of urea may be greatly 
diminished. 

A marked decrease has been observed, also, in dis- 
eases involving structural changes in the tubules of 
the kidney as in parenchymatous nephritis, and this 
suggests again the relation of the kidney to the for- 
mation of urea. 

It has been observed that an increase in the body 
temperature, as from taking hot baths, is followed by 
an increased elimination of urea. This in turn seems 
to be compensated for by a period of diminished elim- 
ination. It is also a matter of practical experience 
that the administration of many inorganic salts causes 
an increase in the quantity of urea passed. The same 
observation has been made with a number of alka- 
loidal salts, but the results of experiments are hardly 
definite or full enough, as yet, to warrant the drawing 
of final conclusions. 

By some authorities ammonium carbonate is looked 
upon as the immediate forerunner of urea, which lat- 
ter is formed by splitting off of water from the former : 

(NH 4 ) 2 C0 3 = CO(NH 2 ) 2 + 2 H 2 0. 



122 URINE ANALYSIS 

As bearing on this it has been observed that relatively 
large quantities of ammonium carbonate and organic 
ammonium salts, when taken as remedies, do not in- 
crease the alkalinity of the urine, but leave the body 
as urea. It has been supposed that this change takes 
place in the liver. 

Recognition of Urea. — Because of its extreme solu- 
bility urea cannot be easily obtained by evaporation 
of urine. It has been shown, however, in an earlier 
chapter that by concentrating the urine slowly to a 
small volume — to one-third or one-fourth — cooling and 
adding strong nitric acid, a crystalline precipitate of 
plates of urea nitrate separates, which is characteristic. 
From this precipitate pure urea can be obtained. 

Clinically, this test has no importance, as we are 
concerned only with a measurement of the amount of 
urea. This determination can be made in several 
ways, but in actual practice we employ three essentially 
different methods. The first depends on the fact that 
solutions of urea precipitate solutions of certain metals 
in a definite manner, from which a volumetric process 
has been derived. The second depends on the fact 
that solutions of certain oxidizing agents decompose 
solutions of urea with the liberation of its nitrogen 
(and carbon dioxide) in gaseous form. From the 
known relations between weight and volume of the 
gas, and weight of nitrogen and weight of urea, the 
absolute amount of the latter may be calculated. The 
third method depends on the fact that when the urea 
of urine is decomposed into water, carbon dioxide and 



UREA 123 

nitrogen, its specific gravity is decreased in a manner 
empirically determined. The loss in specific gravity 
bears a certain relation to weight of urea present. 

Determination of Urea 

Liebig's Method. — We have here the oldest, and, in 
many respects, the best of our processes for the titra- 
tion of urea. The principle involved in the method 
is this : When a solution of mercuric nitrate is added 
to a solution of urea a white precipitate forms and 
settles out. By working with solutions of a certain 
definite concentration it has been found that the reac- 
tion between the mercury and urea takes place in con- 
stant proportion and according to this equation : 

2CON 2 H 4 + 4 Hg (N0 3 ) 2 + 3H 2 = 

2CON 2 H 4 .Hg(N0 3 ) 2 .3HgO + 6HN0 3 

This precipitate contains 10 parts of urea for every 
72 parts of HgO. 72 grams of HgO dissolved in 
HNO should precipitate, therefore, 10 grams of urea. 

The same solution of mercury gives a yellow pre- 
cipitate with solution of sodium carbonate, which is 
used as an indicator in a manner to be described. 

The urea solution to be analyzed is poured into a 
beaker and standard solution of the mercuric nitrate 
added gradually, with constant stirring, from a burette. 
From time to time a drop of the liquid above the pre- 
cipitate is taken on the end of a glass rod and brought 
in contact with a few drops of a concentrated solution 
of sodium carbonate on a plate of dark glass. A yel- 
lowish precipitate forms here if the drop contains any 



124 URINE ANALYSIS 

excess of the mercury compound beyond that neces- 
sary to precipitate the urea. The end of the reaction 
is frequently determined in this manner, as in the 
original process, but not with greatest accuracy. A 
modified process, as now to be explained, is preferable, 
and easily carried out. 

As the equation above shows, nitric acid is set free 
in the reaction between the urea and the mercuric ni- 
trate. * This acid has a decomposing effect on the pre- 
cipitate, tending to form new nitrate and thus dimin- 
ish the amount which, theoretically, should be added 
for complete precipitation. The nitric acid must 
therefore be neutralized from time to time as formed, 
or better, just before the end reaction with the indi- 
cator is tried. 

It has been found, also, that to precipitate exactly 
10 milligrams of urea in this manner, not 72 milli- 
grams of mercuric oxide in solution, but a slightly 
greater amount must be used. The experiments of 
Pflueger showed that 77.2 milligrams is needed for 
the purpose and the standard solution should be made 
to contain 77.2 grains per liter. 

In the titration of urine certain modifications must 
be made which are not necessary in the titration of 
pure urea solutions. The phosphates, sulphates, and 
chlorides of urine interfere with the reaction and must 
be removed before the test is begun. 

The phosphates and sulphates may be removed by 
precipitation with barium solution, while the chlorides 
may be thrown out by silver nitrate. It is also possi- 



UREA 125 

ble to make a correction for the chlorides instead of 
precipitating them. The following solutions are 
necessary in making the test : 

(a) Mercuric Nitrate Solution. — This is made of definite 
strength and should contain the equivalent of 77.2 
grams of the oxide in one liter. In making this 
solution we may start with pure metallic mercury, 
with mercuric oxide or with the commercial ni- 
trate (mercurous). With mercury it can be made 
in this manner : Weigh out a quantity of pure 
mercury and heat it in a porcelain dish or casserole 
with two or three times its weight of strong nitric 
acid of 1.42 specific gravity. When the mercury 
is in solution evaporate to the consistency of a thick 
sirup and add from time to time a few drops of 
nitric acid to complete the oxidation. When the 
addition of the acid is no longer followed by the 
evolution of red fumes the action is complete and 
the mercury exists as mercuric salt. Now pour 
into the sirupy residue ten times its volume of 
water, with constant stirring. In adding the water 
it always happens that a little of the nitrate is de- 
composed and thrown down as a basic salt. Allow 
the liquid to settle thoroughly, pour off from the 
sediment and dissolve the latter in a few drops of 
nitric acid. Add this now to the main solution 
and dilute it with distilled water to make 1 liter 
of each 71.5 grams of mercury. 
When mercuric oxide is employed, weigh out the 
proper amount, dissolve it in a slight excess of strong, 
pure nitric acid, evaporate to a sirup and treat as 
above. Finally, dilute with water to yield a solution 
with 77.2 grams to the liter. 



126 URINE ANALYSIS 

(b) Baryta Solution to precipitate phosphates and sul- 
phates. To one volume of a cold saturated solu- 
tion of barium nitrate add two volumes of a cold 
saturated solution of barium hydroxide. Keep in 
a well-stoppered bottle. 

(V) Sodium Carbonate Solution. — This is best made of 
the pure, dry carbonate readily obtained as a com- 
mercial article. It must be remembered, however, 
that the so-called dry carbonate contains a little 
water, which ma}' be removed by heating, in a 
platinum dish, to low redness. Dissolve 53 grams 
of the salt thus dried, in water, and dilute to one 
liter. 

A mercury solution made of pure material accord- 
ing to the above directions should have the correct 
strength, but for control it may be tested by means of 
a solution of pure urea in water. 

(d) Standard Urea Solution. — Weigh out 2 grams of pure 
urea, which is now readily obtained, and dissolve 
it in distilled water to make 100 cc. 

Before making the test proper it is necessary to de- 
termine the relation of the sodium carbonate solution 
to the mercury solution in presence of urea. This 
may be done by taking exactly 10 cc. of the urea so- 
lution and adding to it 19 cc. of the solution of mer- 
curic nitrate. Shake thoroughly, allow to stand a 
minute and filter. Wash the precipitate with a little 
distilled water, and to the mixed filtrate and washings 
add two drops of a weak solution of methyl orange, or 
enough to give a pink color. Then from a burette 



UREA 127 

run in the solution of sodium carbonate, with constant 
shaking, until the pink color changes to yellow. The 
reaction is sharp enough for the purpose. Not over 
1 1.5 cc. of the alkali solution should be required for 
this. Calculate the amount needed for each cubic 
centimeter of mercuric nitrate. Proceed now with the 
actual test. 

Measure 10 cc. of the standard urea solution again 
and run in 19.5 cc. of the mercuric nitrate. Add now 
the correct number of cubic centimeters of soda solu- 
tion required to neutralize the acid from the nitrate, 
as calculated from the results of the last experiment. 
Then by means of a stirring rod bring a drop of the 
liquid in the beaker in contact with a drop of a 
semifluid mixture of sodium bicarbonate and water on 
a dark glass plate. Stir the two together and observe 
the color. It should be white. Run in two or three 
drops more of the mercury solution, stir well and re- 
peat the test, and continue until a slight yellow color 
is obtained on mixing the drops from the beaker with 
the moist bicarbonate. If the mercury solution is 
correct, just 20 cc. should be used for this. 

The sodium bicarbonate used as indicator must be 
pure, especially as regards freedom from chloride. An 
excess of it can be washed in a beaker several times 
with a little cold water, which is poured off leaving 
the salt finally in a pasty condition suitable for use. 

We proceed now to the actual test of a sample of 
urine. Measure off accurately a definite volume and 
add to it just half its volume of the baryta solution. 
Convenient proportions are 50 cc. of urine and 25 cc. 



128 URINE ANALYSIS 

of the baryta solution. Shake thoroughly and filter 
through a dry filter into a flask. Measure out now 
exactly 15 ee. of this filtrate which contains the urea 
of 10 cc. of the original urine, the baryta solution hav- 
ing taken out only phosphates, sulphates, and carbon- 
ates, with certain bases. This filtrate still contains 
chlorides which are objectionable and which could be 
separated by another precipitation with the proper 
amount of silver nitrate. It will be, however, more 
convenient, and fully as accurate, to make a correction 
for them at the end of the test, as will be explained. 
The 15 cc. of filtrate has an alkaline reaction and must 
be neutralized. (If not alkaline a new precipitation 
must be made, taking equal volumes of urine and 
baryta solution, and finally 20 cc. of the filtrate.) The 
neutralization can be effected by adding carefully, a 
drop at a time, dilute nitric acid, testing with litmus 
paper. 

The filtrate thus prepared is titrated with the mer- 
cury solution. Begin by adding a cubic centimeter 
at a time and after each addition bring a drop of the 
mixture in contact with a drop of the semifluid sodium 
bicarbonate on a plate of dark glass. The drops 
should be placed side by side and mixed at the edges. 
At first the mixture remains white, even after stirring, 
but as the addition of mercury is continued a point is 
reached where the drop from the beaker brought in 
contact with the moist bicarbonate gives a light yel- 
low shade. On stirring the drops together this yel- 
low should disappear, but this shows that the end of 
the reaction is nearly reached. Add now the mercury 



UREA 1 29 

solution in drops and test after each addition. When 
the point is reached where a faint yellow shade per- 
sists after stirring together the drop from the beaker 
and the sodium bicarbonate, it is time to neutralize 
with the normal sodium carbonate solution. Run in 
the right number of cubic centimeters corresponding 
to the mercury used and now make the test for the 
final reaction again and continue until the yellow color 
appears. 

Regard this test as preliminary and make a new 
one w r ith 15 cc. of the filtrate neutralized as before. 
Run in directly within 1 cc. of the amount of mercury 
required, as shown by the first test, neutralize and 
complete as before. For each cubic centimeter used, 
after deducting for chlorides, calculate 10 milligrams 
of urea. The correction for the chlorides is based on 
the following principle : In the presence of the sodium 
chloride, or other chloride, the reaction between mer- 
curic nitrate and urea does not begin until enough of 
the former has been added to form mercuric chloride 
with all chlorine present, according to the following 
equation : 

Hg(N0 3 ) 2 + 2NaCl = 2NaN0 3 + HgCl,. 

For the nitrate corresponding to 216 parts of HgO 
we use here 117 parts of salt, or for 117 milligrams of 
salt, 216 milligrams of mercuric oxide, or 2.79 cc. of 
the standard mercuric nitrate solution. 

One milligram of sodium chloride, therefore, com- 
bines with the mercury compound in 0.0238 cc. of the 
standard solution. Mercuric chloride does not react 



130 URINE ANALYSIS 

with the sodium bicarbonate, and the amount formed 
is beyond that shown by the titration. Therefore, to 
apply the correction, determine the chlorides present 
in 10 cc. of urine (by a process to be given later), cal- 
culate to sodium chloride, and for each milligram 
found deduct 0.0238 cc. from the volume of the mer- 
curic nitrate solution used in the titration. As the 
amount of chlorine in the urine is about equivalent to 
1 per cent, of salt, in the mean, an approximate cor- 
rection is often made by subtracting 2 cc. from the 
volume of the mercury solution. 

The above calculations are based on the supposition 
that the original urine contains 2 per cent, of urea, 
and that a volume of about 20 cc. of mercuric nitrate 
is used in the titration. If the per cent, of urea is 
much greater or less than this, a correction on account 
of volume must be made. This correction has been 
worked out empirically by Pflueger, and, without dis- 
cussing how it is derived, it will be sufficient to ex- 
plain its application. 

If more than 2 per cent, of urea is present it is nec- 
essary to add more than 20 cc. of the mercuric solu- 
tion in titration. If the volume of the latter solution 
is greater than the sum of the volumes of the prepared 
urine and soda solution used in neutralization, this 
sum must be subtracted from the volume of the mer- 
cury solution and the result multiplied by 0.08. The 
product is added to the number of cubic centimeters 
of mercuric nitrate used, to give the corrected result. 
If, on the other hand, the volume of mercuric nitrate 
used in titration is less than the sum of the volumes 



UREA 131 

of prepared urine and soda solution, the difference is 
multiplied by 0.08 and the product taken from the 
number of cubic centimeters of mercuric solution used, 
to give the corrected result. In these calculations the 
volume of mercuric nitrate taken up by the chlorides 
must be considered as part of the diluting liquid. The 
same must be remembered in adding sodium carbon- 
ate for neutralization. 

The correction may be expressed in this formula, 
according to Pflueger : 

C = (V, — VJ X 0.08, 
in which 

C = the correction to be added or subtracted ; 

Vj = the sum of the volumes of the urine, soda solu- 
tion and mercuric nitrate combined with the 
chlorides ; 

V a = the volume of mercuric nitrate taken by urea. 

In illustration we may take an actual case : 

15.0 cc. = the prepared urine (neutralized); 
15.8 cc. = the sodium carbonate used ; 
1.8 cc. = the mercuric solution used by chlorides. 



Vj =32.6 cc. 

V» — 26.0 cc. 



6.6 

(\\ — VJ X 0.08 = 0.528 = c. 

Therefore, 26 — 0.5 = 25.5 is the corrected vol- 
ume of mercuric nitrate, indicating, with the latter 
solution of the standard strength, 25.5 grams of urea 
in a liter. 



132 URINE ANALYSIS 

The results of the Liebig method are usually a little 
high because urine contains, besides urea, several other 
substances which react with the mercuric nitrate. The 
first of these is ammonia, which is generally present 
in small amount in normal fresh urine and often in 
larger amount in pathological urines. A process for 
the determination of the ammonia w r ill be given later. 
It has been found that 10 milligrams of NH require 
about 2.0 cc. of the standard mercuric solution and on 
this basis a correction may be made. In normal urine 
the correction would amount to about 1.0 cc. of the 
mercuric solution, in the mean, for the volume of 
filtrate used. 

Uric acid, hippuric acid, creatin, creatinin and 
traces of other bodies containing nitrogen, have also a 
disturbing action and make the calculated per cent, of 
urea appear higher than it should be. A slight 
correction should be therefore introduced here too. 
Taking all the nitrogenous bodies, including ammonia, 
into consideration, it is safe to subtract 2 cc. from the 
volume of the mercuric solution used to find that ac- 
tually required by the urea. 

If the urine contains albumin this must be coagu- 
lated and filtered out. To accomplish this, measure 
50 cc. of the urine, add a few drops of acetic acid and 
boil to completely coagulate. Allow to cool, filter 
and add a little water to compensate for that lost by 
evaporation in the boiling. With the filtrate proceed 
as before. 

From what has been said it will be recognized that 
the Liebig process is one for the determination of the 



UREA 133 

total nitrogenous matter in urine rather than for the 
titration of urea alone. Because of the several correc- 
tions which must be applied to the results as obtained, 
many chemists prefer to employ a different process, 
depending on an entirely different principle, which 
will now be explained. 

Method by Liberation of Nitrogen. — A solution of 
urea is decomposed by a solution of a hypochlorite or 
hypobromite as illustrated by this equation. 
CON,H 4 + 3NaOCl = C0 2 + N 9 + 2 H 2 + 3 NaCl. 

That is, nitrogen and carbon dioxide gases are given 
off. If the reaction is allowed to take place in an al- 
kaline medium the carbon dioxide will be held and 
the nitrogen alone given off. The volume liberated 
is a measure of the weight of urea decomposed. From 
the above equation it is seen that 28 parts by weight 
of nitrogen correspond to 60 of urea, from which it fol- 
lows that 1 cc. of pure nitrogen gas, measured at a 
temperature of o° C. and under the normal pressure 
of 760 mm. corresponds to 0.00269 gram of urea. One 
gram of urea furnishes 371.4 cc. of nitrogen gas. 

In employing these principles in practice 'it is sim- 
ply necessary to bring together a measured volume of 
the urine or urea solution and the hypochlorite or 
hypobromite reagent under such conditions that all of 
the nitrogen liberated may be collected and accurately 
measured. 

As a reagent a solution of sodium hypobromite is 
very commonly employed. As it does not keep well 
it must be made fresh for use, which is inconvenient 



134 URINE ANALYSIS 

unless many tests have to be made at one time. The 
reagent may be prepared in this manner: 

Dissolve i oo grams of good sodium hydroxide in 250 
cc. of water. When cold add 25 cc. of bromine by 
means of a funnel tube carried to the center of the 
solution. The bromine must be poured into the fun- 
nel, a little at a time, and the lower end moved around 
to act as a stirrer and mix the liquids. During the 
reaction the bottle or flask containing the alkali should 
be surrounded by cold water, and the mixture should 
be made out of doors or in a good fume closet. The 
finished solution contains an excess of alkali sufficient 
to hold the carbon dioxide given off when it reacts on 
urea. In place of this solution a strong hypochlorite 
solution may be used with advantage. The U. S. P. 
solution when properly made answers very well. Its 
preparation is given in the appendix. 

Many forms of apparatus have been devised for this 
purpose, one of the oldest and best known of which is 
that of Huefner, shown in Fig. 3. 

At A ) below, is a small vessel, the bottom of which 
rests on the support, and which holds the urine to be 
decomposed. The capacity of this vessel is usually 
between 5 and 10 cc, but must be accurately deter- 
mined by filling with mercury, pouring this out and 
weighing it. Above A, and separated from it by a 
ground glass stop-cock, is the much larger vessel B y 
which holds the hypochlorite or hypobromite reagent. 
On the narrow neck of B rests a cup-shaped recepta- 
cle, C, to hold water. Finally, over B a measuring 
tube is supported in such a manner that it must re- 



UREA 

ceive any gas passing up from B. 
of the experiment this 
measuring tube D is filled 
with water. 

The apparatus is used 
in the following manner : 
Rinse out A and B and 
pour into the latter more 
than enough urine to fill 
A. Open the stop-cock 
and allow the urine to 
flow down, which may be 
assisted, when slow, by 
moving a thin glass rod 
or bit of wire up and down 
through the opening in the 
stopper. When A is quite 
full, close the stopper, 
rinse the urine from B 
and fill it with the hypo- 
bromite reagent. Now fill 
the cup C with water so 
that its surface is one or 
two centimeters above the 
opening into B, fill the 
graduated tube with water, 
close the end with the 
thumb and invert it in the 
cup. Finally fasten it in 
position over B as shown. 



135 
At the beginning 




The stop-cock is now opened which permits the 



36 



URINE ANALYSIS 



heavier reagent to sink and slowly mix with the urine. 
A liberation of gas soon begins and proceeds slowly. 
At the end of twenty or thirty minutes the reaction is 
complete, which can be noted by the disappearance of 
the gas bubbles. Close the end of the gas tube with 
the thumb, remove it and immerse in a jar of 
w 7 ater, having the temperature of the air, until the 
water levels inside the tube and in the jar, are the 
same. Clamp the tube in position and allow it to 
stand until the temperature of the gas becomes con- 
stant, which may require fifteen minutes. Finally 
adjust the tube level again, if necessary, read the vol- 
ume of the gas, note the height of the barometer and 
the temperature, as given by a thermometer hanging 
near the tube, and reduce this volume to standard con- 
ditions by the following formula : 



Y c = V 
In this formula 



b 



(i -f 0.00366^) 760 



V c = the corrected volume ; 
V = the observed volume ; 
b = the barometric height ; 

w -- the tension of water vapor at the observed tempera- 
ture, in millimeters of mercury ; 
t = the temperature as observed. 

The values for w at different temperatures are given 

in this table : 



t 


w 


t 


w 


t 


w 


o° •• 


• 9.165 mm. 


16 


.. 13.536 mm. 


21° • 


. 18.495 mm. 


1° .. 


. 9.792 mm. 


17° 


. . 14.421 mm. 


22° • 


• 19-659 nun. 


2° -. 


. 10.457 mm - 


18 


.. 15.357 mm. 


23° • 


. 20.888 mm. 


3°-- 


. 1 1. 162 mm. 


19° 


• . 16.346 mm. 


24°.. 


. 22.184 mm - 


4 .. 


. ir. 908 mm. 


20° 


• • 17.391 mm. 


25° •• 


- 23.550 mm. 


5°-- 


• 12.699 mm - 











UREA 



137 



Having thus the volume of the gas under normal 
conditions, the weight of urea corresponding can be 
calculated by data already given. 

The results obtained by this method are too low and 
must be corrected, as will be explained later. 

Another form of urea 
apparatus which can be 
constructed by any one is 
shown in Fig. 4. 

The tall jar is filled 
with water which must 
stand until it has the air 
temperature. A 50 cc. 
burette is inverted in the 
j ar, the delivery end being 
connected with a bottle 
holding about 150 cc, by 
means of a piece of firm 
rubber tubing. The rub- 
ber tube is slipped over a 
short glass tube passing 
through the hole in a rub- 
ber stopper which must 
close the bottle accurately. 
In the bottle is a short 
stout test-tube, or vial, p^ 4 

holding about 10 cc. and 

which contains the urine to be tested. Into the bottle 
itself is poured the reagent as above described, which 
must not reach to the top of the test-tube. On mixing 
the liquids the urea decomposes, liberating the gas as 




138 URINE ANALYSIS 

before, which passes through the rubber tube and 
displaces water in the burette so that the volume can 
be readily determined. 

The test is made practically in this manner : Pour 
about 20 cc. of the strong hypobromite or 40 cc. of the 
weaker hypochlorite reagent into the bottle. With a 
pipette measure some exact volume of urine, usually 
5 cc, into the small test-tube, and by means of small 
iron forceps place the latter carefully in the bottle 
containing the reagent. Next insert the stopper which 
connects the bottle with the burette standing in the 
jar of water. The level of the water in the burette is 
displaced by this operation. Now allow the appara- 
tus to remain at rest ten minutes until the air in the 
mixing bottle and tube has the temperature of the 
outside air. Through handling, it of course becomes 
warmer. At the end of the time, by means of the at- 
tached clamp, and not by the hand, lift the burette, or 
depress it, if necessary, until the water levels inside 
and outside are the same. Note the position of the 
water on the burette graduation. Read the air temper- 
ature by a thermometer, which should be suspended 
near the tube, and observe the height of the barometer. 

Incline the bottle to mix the urine and the reagent, 
shake gently and repeat these operations from time to 
time. On the decomposition of the urea nitrogen gas, 
or its equivalent volume of air, passes over into the 
burette and forces out some of the water. When, after 
repeatedly shaking the bottle, no increase of the gas 
volume in the burette can be observed, allow the whole 
apparatus to stand until the contents of the bottle and 



UREA 139 

burette have cooled down to the air temperature again. 
Then lift the burette, with the clamp as before, to re- 
store the levels and read the gas volume. From this 
subtract the volume at the first reading. The differ- 
ence is the volume of nitrogen gas liberated in the re- 
action, at the observed temperature and atmospheric 
pressure. If, as sometimes happens, more gas is lib- 
erated than the burette will hold, repeat the experi- 
ment, using urine diluted with an equal volume of 
water. 

The calculations are made as in the case given 
above, as the gas volume is finally measured under the 
same conditions. It is assumed here that the air tem- 
perature and barometric pressure remain constant dur- 
ing the experiment. 

Exact investigations have shown that the whole of 
the nitrogen is not liberated in the reaction, as at first 
assumed, but falls short between 7 and 8 per cent. It 
appears that under some conditions a small part, pos- 
sibly 3 or 4 per cent., of the nitrogen of the urea is 
oxidized to nitric acid in the reaction and escapes 
measurement. Another small portion is left in the 
ammoniacal condition. Attempts have been made to 
prevent this abnormal oxidation by adding to the urine 
a reducing agent to destroy nitric acid as fast as formed. 
Dextrose has been used for the purpose, also cane- 
sugar, and apparently with success. But a great ex- 
cess of sugar must be added. 

It has been shown, also, that the theoretical yield of 
nitrogen can be approached by a modified method of 
applying the reagent. J. R. Duggan, working in 



140 URINE ANALYSIS 

Remsen's laboratory, found that by mixing the alkali 
with the urine and then adding the bromine so as to 
form hypobromite in presence of the urea, the yield of 
nitrogen is much increased, reaching nearly the amount 
called for by theory. The simple bottle and burette 
apparatus may be used in this manner by measuring 
the alkali, 20 cc. of 20 per cent, sodium hydroxide 
solution, and urine, 5 cc.,into the bottle, and the bro- 
mine, about 1 cc, into the test-tube. The mixture is 
made and process completed as before. The modifica- 
tion has not come into general use, probably because 
of the objection to working with free bromine, at each 
test. As the deficiency by the usual method has been 
shown to be nearly 8 per cent., it is sufficient for most 
purposes to assume that the volume of gas obtained is 
92 per cent, of the whole and correct by calculation. 

It should also be stated here that there are certain 
positive errors in the process as in that of Liebig. The 
hypobromite acts not only on urea, but on uric acid, 
ammonia and on other normal urine constituents. In 
most of these cases not all of the nitrogen present is 
given off in the free state, but the fraction which is 
liberated is enough to cause a very sensible error in 
the process. It is generally overlooked because the 
opposite or minus error is so much greater. Attempts 
are sometimes made to remove these disturbing sub- 
stances by precipitation with phosphotungstic acid 
before applying the Liebig or hypobromite process, 
but in every-day clinical practice this complication 
cannot be recommended. 



UREA 



141 



It remains now to describe two forms of apparatus 
which are used without any correction, for rapid clin- 
ical tests. 

The Squibb Apparatus is the first of these, and one 
which can be very highly recommended. The con- 
struction of the apparatus is shown by Fig. 5. 

The upright bottle to the left contains the reagent, 
hypochlorite or hypobromite as before. By means of 
a pair of small forceps a short test-tube, F, containing 
a measured volume of the urine is dropped into the 
bottle, but carefully, so that the liquids will not mix 




until the bottle is shaken. A bent glass tube and a 
rubber tube connect this with the bottle B, which at 
the beginning of the test is quite filled with water. 
Another glass tube connects B with the bottle Z>, 
empty at the beginning of the test. 

To make the test pour into the first bottle 20 cc. of 
strong hypobromite solution, or 40 cc. of the hypo- 
chlorite. Measure accurately 4 or 5 cc. of urine into 



142 URINE ANALYSIS 

the tube, drop this into the bottle and insert the stop- 
per. Fill B quite full of water, and insert its stopper 
which drives out a little water through the short tube 
into D. Allow the whole apparatus to stand ten min- 
utes to take the temperature of the air, then empty D 
and replace it. Now tip the first bottle so as to mix 
the contents of F with the reagent and shake gently. 
Bubbles of gas escape, and, passing over into B, drive 
out a corresponding volume of water. Repeat the 
shaking- of the reagent bottle several times. In a few 
minutes the reaction is complete, but the apparatus 
must be allowed to stand to cool down to the air tem- 
perature. A part of the water in D may be drawn 
back into B. Finally measure the volume of water 
left in D, and take this as the volume of gas liberated. 
Make the calculation as before on the assumption that 
each cubic centimeter of gas corresponds to 0.002 7 gram 
of urea. In this there are two errors which nearly com- 
pensate each other. In the first place not all the gas 
is liberated for the reasons explained above, but in the 
second place the volume read off is higher than nor- 
mal because of higher temperature and lower barome- 
ter. The results obtained may be, therefore, nearly 
correct, sufficiently so for all clinical purposes, through 
compensation of errors. 

Squibb has constructed a table from which the per- 
centage of urea corresponding to any volume of gas, 
for a given volume of urine taken, can be read at a 
glance without any calculation whatever. This is 
convenient, but not necessarv. 



UREA 143 

The Doremus Apparatus. — This is shown in the an- 

Onexed cut, and at the present time is very 
widely used by physicians because of the 
simplicity of the method of employing it. 
The graduated tube is filled to an indi- 
cating mark with strong hypobromite so- 
lution and then water is added to fill the 
remainder of the tube and the bulb. By 
S means of the pipette, graduated to hold 1 
J cc, this quantity of urine is forced into 
S the liquid in the upright part of the tube. 
Fig. 6. The urea is immediately decomposed, and 
its nitrogen ascends to the top of the graduated part, 
where it is read off. The longer marks on the tube 
indicate the fraction of a gram of urea in the 1 cc. of 
urine taken ; the shorter marks indicate tenths. Mul- 
tiplying by 100 we obtain the number of grams of 
urea in 100 cc. The results are apt to be low from a 
loss of nitrogen through the bulb, which can scarcely 
be avoided. The instrument is said to be "good 
enough" for clinical purposes, but cannot be com- 
pared with that of Squibb for accuracy. No instru- 
ment in which the volume of urine taken for the test 
is as small as 1 cc. should be expected to give even 
approximately accurate results, unless very great pre- 
cautions are taken in the measurement and in subse- 
quent parts of the work. 

The Total Nitrogen. — By this is understood the nitro- 
gen of all the excreted urinary products. It is approxi- 
mately measured by the Liebig process, uncorrected, 



144 URINE ANALYSIS 

and may be determined with perfect accuracy by sev- 
eral methods. The Kjeldahl process is most conve- 
niently applied but, like all the others, in it too many 
manipulations are required to make it available for 
clinical purposes. For a description of the details of 
the Kjeldahl process the reader is referred to special 
works on organic analysis. 



Chapter VII 

THE DETERHINATION OF PHOSPHATES, CHLO- 
RIDES, AND SULPHATES 

Phosphates 

Phosphoric acid occurs normally in the urine com- 
bined with alkali and alkali-earth metals, of which 
combinations the alkali phosphates are soluble in 
water, while the earthy phosphates are insoluble. In 
the urine, how T ever, they are held in solution through 
several agencies. The larger part of the earthy phos- 
phates appear to be held here normally in the acid 
condition ; that is, as compounds of the formulas 
CaH 4 (P0 4 ) 2 and MgH 4 (P0 4 ) 2 . The salts of the type 
CaHPO are present, also, in small amount. As long 
as the urine maintains its acid reaction these bodies 
may be expected to remain in solution, but if it be- 
comes alkaline by fermentation, or by the addition of 
the hydroxides or carbonates of ammonium, sodium, 
or potassium, the acid phosphates are converted into 
insoluble, neutral phosphates and precipitated. Most 
urines contain along with the acid phosphates traces of 
neutral phosphates which precipitate on boiling. It 
has been suggested that these phosphates are held by 
traces of ammonium compounds or by carbonic acid, 
both of which are driven off by heat, allowing the 
phosphates to precipitate. It is well known, how- 



I46 URINE ANALYSIS 

ever, that some urines can be boiled without showing 
any sign of precipitation. In such cases it is proba- 
ble that the neutral phosphates are not present. 

Part of the phosphoric acid of the urine comes di- 
rectly from the phosphates of the food and another 
portion results from the oxidation of the phosphorus- 
holding tissues. In health, the rate of such oxidation 
is practically constant, or nearly so, but in disease it 
may be greatly increased or diminished. Variations 
in the amount of excreted phosphates may become, 
therefore, of considerable clinical importance. Great 
care must be observed, however, in drawing conclu- 
sions regarding the destruction of phosphatic tissues 
from the results obtained by analysis, because of the 
uncertainty of the amount taken with the food and of 
what must be considered a normal excretion of phos- 
phoric acid. 

Wheat bread constitutes one of our important arti- 
cles of food containing a relatively high amount of 
phosphates. Owing to changes in milling processes 
introduced and extended in the past twenty years, very 
material reductions have been made in the percentage 
of phosphates and other mineral substances left in our 
fine flour. The resulting diminution in the phosphates 
of the urine from this cause is appreciable. It must 
be remembered also that a part of the phosphoric acid 
taken with the food is eliminated in insoluble form 
with the feces. The amount so disposed of depends 
on the nature of the original condition of combination 
of the phosphoric acid and on the amount of alkali- 
earth bases present. These tend to form insoluble 



THE DETERMINATION OF PHOSPHATES, ETC. 147 

phosphates. With an exclusively vegetable diet the 
phosphates would, for this reason, be low, while with 
a meat diet more would be excreted, because here the 
alkali phosphates, especially potassium phosphate, are 
in excess. On the other hand the phosphates are 
greatly increased in the urine of people who consume 
great quantities of the various phosphatic beverages 
which have become popular in the United States in 
the past few years. It is readily seen how quite er- 
roneous conclusions could be drawn from the tests of 
urine of such persons. Single analyses for phosphoric 
acid may be very misleading. When the amount of 
phosphoric acid passed with a given diet is known, 
variations observed may sometimes be traced to cer- 
tain pathological conditions briefly mentioned in the 
following paragraph. 

In disease, phosphates are found increased in rickets 
and osteomalacia, possibly from the failure to deposit 
the earthy phosphates in the bones. Meningitis is 
accompanied by an increase of the phosphates. The 
same is true of other disorders of the brain as this or- 
gan is especially rich in phosphatic substances. It is 
said that phosphates are increased after a period of 
severe nervous strain. What has been termed phos- 
phatic diabetes has been described as a condition in 
which a persistent excretion of relatively large amounts 
of the phosphates is observed. The earthy phosphates 
so discharged may amount to 20 grams or more in a 
day. The causes leading to this condition are not 
clearly defined. 



I48 URINE ANALYSIS 

The phosphates have been found diminished in dis- 
eases accompanied by diminished nutrition, and in 
some diseases of the heart and kidney. In dilute urine, 
low specific gravity and high volume, the percentage 
amount of phosphoric acid is much decreased, but it 
does not follow that a decrease in the amount excreted 
in the twenty-four hours must also be small. A safe 
estimate can be made only with a mixed specimen 
taken from the urine of the whole day with considera- 
tion of the volume passed. 

Various statements are found in the books regarding 
the mean excretion of the alkali and earthy phos- 
phates. Different observers have reported between 2 
and 5 grams of phosphoric anhydride (P 2 0_), while 3 
grams may be taken, perhaps, as the mean. 

The recognition of the phosphates is an extremely 
easy matter. The presence of earthy phosphates may 
be shown by adding to the urine enough ammonia 
water to give a faint alkaline reaction and then warm- 
ing. A flocculent precipitate, resembling albumin, 
appears and is usually white, or nearly so. But some- 
times coloring-matters come down with it in amount 
sufficient to give it a brownish or reddish shade. It 
will be recalled that the color of this precipitate was 
referred to under the head of blood tests. 

The alkali phosphates can be detected in the filtrate 
after separation of the earthy phosphates. To this 
end, add to the clear alkaline liquid a little more am- 
monia and some clear magnesia mixture. A fine crys- 
talline precipitate of ammonium-magnesium phosphate 
separates and settles rapidly. This is very character- 



THE DETERMINATION OF PHOSPHATES, ETC. 1 49 

istic. The qualitative tests for phosphates have, how- 
ever, little value in examination of the urine. We 
are chiefly concerned with the amount, the measure- 
ment of which will now be described. 

Determination of Phosphates. — It is customary to 
measure the total phosphoric acid, not the alkali or 
earthy phosphates, separately. We have at our dis- 
posal several methods, gravimetric and volumetric, of 
which the latter are accurate and most convenient. A 
volumetric process will be described which serves for 
the measurement of the phosphoric acid as a whole, 
and which can be used for the separate measurement 
of the earthy and alkali phosphates by dealing with 
the precipitate and filtrate described in the qualitative 
test above. This method depends on the fact that so- 
lutions of uranium nitrate or acetate precipitate phos- 
phates in greenish-yellow colored, flocculent form, and 
that in a solution holding in suspension a precipitate 
of uranium phosphate any excess of soluble uranium 
compound may be recognized by the reddish brown 
precipitate which it gives with a solution of potassium 
ferrocyanide. The latter substance serves, therefore, 
as an indicator. If to a phosphate solution in a beaker 
a dilute uranium solution be added precipitation con- 
tinues until the whole of the phosphates have gone 
into combination with the uranium. If, during the 
precipitation, drops of liquid from the beaker are 
brought in contact with drops of fresh ferrocyanide 
solution on a glass plate, no reddish brown precipitate 
of uranium ferrocyanide appears until the last trace of 
uranium phosphate has been formed. The produc- 



150 URINE ANALYSIS 

tion of uranium ferrocyanide is the indication, there- 
fore, of the finished precipitation of the phosphates. 

The reaction between uranium and phosphates in 
acetic acid solution is illustrated by this equation : 
U0 2 (N0 3 ) 2 + KH 2 P0 4 = UO,HP0 4 - KNO s + HN0 3 

From this it appears that 239.6 parts of uranium 
are required to precipitate 71 parts of P 2 0_. In order 
to have the reaction take place as represented above, 
it is necessary to neutralize the liberated nitric acid as 
fast as formed or dispose of it in some other manner. 
The best plan is to add to the solution to be precipi- 
tated acetic acid and sodium acetate, the first of which 
brings the phosphates into the acid condition, while 
the second is decomposed with the formation of sodium 
nitrate and free acetic acid. Mineral acids interfere 
with the reaction, while moderate amounts of acetic 
acid do not. 

In order to carry out this method we prepare the 
following solutions : 

{a) Standard Uranium Solution. — This is made by dis- 
solving 36 grams of the pure crystallized nitrate, 
U0 2 (NOJ 2 6H 2 0, in distilled water to make one 
liter. The strength of the solution is adjusted by 
experiment as explained below. 

(b) Standard Phosphate Solution. — This is made by dis- 
solving 10.085 grams of pure crystals of sodium 
phosphate, HNa 2 P0 4 . i2H,0, in distilled water to 
make 1 liter. 50 cc. of this solution contains o. 100 
gram of P 2 5 . Small fresh, unefnoresced crystals 
of the phosphate must be used for this solution. 



THE DETERMINATION OF PHOSPHATES, ETC. 151 

(c) Sodium Acetate Solution. — Dissolve 100 grams in 
800 cc. of distilled water, add 100 cc. of 30 per 
cent, acetic acid and then water enough to make 1 
liter. 

(d) Fresh Ferrocyanide Solution. — Dissolve 10 grains of 
pure potassium ferrocyanide in 100 ce. of distilled 
water. The solution should be kept in the dark. 

The actual value of the uranium solution is deter- 
mined by the following experiment : Measure out 50 
cc. of the phosphate solution, {b\ add 5 cc. of the ace- 
tate solution, (V), and heat in a beaker in a water-bath 
to near the boiling temperature. Place several drops 
of the ferrocyanide solution on a white plate. Fill a 
burette with the uranium solution and when the solu- 
tion in the beaker has reached the proper temperature 
run into it from the burette 18 cc. of the uranium 
standard. Warm again, and by means of a glass rod 
bring a drop of the liquid in the beaker in contact 
with one of the ferrocyanide drops on the plate. If 
the uranium solution has been properly made no red 
color should yet appear. Now run in a fifth of 1 cc. 
more from the burette, warm and test again, and re- 
peat these operations until the first faint reddish shade 
begins to show on bringing the two drops in contact. 
With this test as a preliminary one make a second, 
adding at first one-fifth of a cubic centimeter less than 
the final result of the preliminary, and finish as be- 
fore. Something less than 20 cc. should be needed 
to complete the reaction. Supposing 19.8 cc. are re- 
quired for the purpose, the whole solution should be 
diluted in the proportion, 

19.8 : 20 : : a : x 



152 URINE ANALYSIS 

in which a represents the volume on hand. We have 
now a standard uranium solution, each cubic centi- 
meter of which precipitates exactly 5 milligrams of 
P 2 , and with this we are able to measure phosphoric 
acid in unknown solutions. 

It is sometimes recommended to use uranium ace- 
tate instead of nitrate in making this standard solu- 
tion, and so avoid a disturbing element in the libera- 
tion of the nitric acid. But there are several advan- 
tages in the use of the nitrate which should be men- 
tioned. In the first place its solutions keep better, 
and secondly, it can be obtained in commerce in almost 
(if not quite) chemically pure condition, so that it is 
possible to make up a solution of nearly correct strength 
by simply weighing out and dissolving the crystals. 
Assuming 239.6 as the atomic weight of uranium, and 
O = 16, the relation of the crystallized pure nitrate 
to P 2 0. is 

1007.2 : 142, 

from which it follows that a liter of the standard solu- 
tion should contain 35.46 grains of the uranium salt, 
if each cubic centimeter is to indicate 5 milligrams of 

PO. 

2 5 
The solution of sodium acetate with acetic acid must 

always be added in the proportion given above if uni- 
form results are expected, and the ferrocyanide indi- 
cator must be fresh and as weak as given. 

The test of urine is made exactly as given in the 
above. Measure out 50 cc. of urine, add 5 cc. of the 
acetate mixture, and finish as before. The 50 cc. of 
urine, in the mean, contains about as much phosphoric 



THE DETERMINATION OF PHOSPHATES, ETC. 1 53 

acid as was present in the same volume of standard 
phosphate solution. The titration must be made hot, 
because the reaction is much quicker and sharper in 
hot solution than in cold. Make always two tests ; 
the first is an approximation, while the second gives 
a much closer result. 

A separate test of the earthy phosphates may be 
made by adding to 200 cc. of urine enough ammonia 
to give an alkaline reaction. The urine then must 
stand until the precipitated phosphates settle out. The 
precipitate is collected on a small filter, washed with 
water containing a very little ammonia, and then 
allowed to drain. It is next dissolved in a small 
amount of acetic acid, the solution diluted to 50 cc, 
mixed with 5 cc. of the sodium acetate solution and 
titrated as before. The reaction here is not quite as 
accurate as with the alkali phosphate, but the results 
are satisfactory for the purpose. The difference be- 
tween the total phosphates and the earthy phosphates, 
expressed in terms of P 2 , is the amount combined 
as alkali phosphates. 

It is also possible to determine the amount of phos- 
phoric acid combined as monohydrogen salt and that 
combined as dihydrogen salt, but the determination 
has at the present time little clinical value. 

Instead of finding the end point in the precipitation 
with uranium solution by means of drops of ferrocya- 
nide as explained, the following process may be fol- 
lowed. Add to the urine the sodium acetate as before 
and then three or four drops of cochineal solution, 
made as given in the appendix. Heat to boiling and 



154 URINE ANALYSIS 

add the uranium solution to the hot liquid. Just as 
soon as the phosphate is combined and a trace of ura- 
nium left in excess, it produces a green color or pre- 
cipitate with the cochineal which thus serves as an 
indicator to show the end of the reaction. If the 
urine is quite warm the color is sharp and can be 
quickly seen so that the reaction may be considered a 
sensitive one. The same test may, of course, be used 
to standardize the uranium solution at the outset. 

Chlorides 

Practically, all of the chlorine taken into the stom- 
ach w T ith the food is eliminated with the urine. The 
chlorine entering the body is mostly in the form of 
sodium chloride, although traces of other chlorides are 
found in some of our foodstuffs. 

The amount of salt excreted depends, therefore, 
closely on that consumed, and varies within wide 
limits. It is commonly said that 10 to 15 grams daily 
include the amounts passed in the great majority of 
urines. It must be remembered, however, that in in- 
dividual cases the upper limit may be very greatly 
exceeded. In the urine of men who. eat a great deal 
of salt food, from 20 to 25 grams are frequently found. 
In such cases the volume of water drunk is usually 
large, so that the percentage amount of salt passed 
does not follow 7 the same variations. Expressed in 
this manner, about 1 per cent, represents the average 
excretion. 

Pathologically, chlorides are increased in total 
amount in diabetes insipidus, and temporarily, some- 



THE DETERMINATION OF PHOSPHATES, ETC. 1 55 

times in intermittent fevers. A decrease in the chlo- 
rides is more frequently observed, and has greater 
clinical importance. 

This decrease is noticed generally in febrile condi- 
tions, and especially if salty exudations are being 
formed in any part of the body. In the serous accu- 
mulations of pleurisy common salt is abundant, and 
at the same time greatly diminished in the urine. The 
expectorated fluid in cases of acute pneumonia con- 
tains much salt, which in consequence is decreased in 
the urine. In some cases the chlorine is practically 
absent from the urine. In any event its reappearance 
in normal amount during the progress of disease is a 
favorable sign, as indicating the approach of normal 
conditions of absorption and excretion in the body. 
Quantitative tests for the chlorides of the urine be- 
come, therefore, of great importance, as their absence 
or marked decrease can only occur in disorders of seri- 
ous nature. Fortunately, such tests are very easily 
made, and by simple volumetric processes. 

Volumetric Determination of Chlorine. — The best pro- 
cesses by which chlorine is measured volumetrically 
in the urine and elsewhere depend on the reaction be- 
tween chlorides and silver nitrate, 

NaCl -f AgN0 3 = AgCl + NaN0 3 , 
from which it appears that 5.85 milligrams of salt re- 
quire for precipitation 16.99 milligrams of silver ni- 
trate. In many cases, if a weak solution of silver ni- 
trate be added to a weak solution of salt in a bottle, 
with frequent shaking, the curdy precipitate of silver 



156 URINE ANALYSIS 

chloride formed will settle out so rapidly that it is 
possible to determine just when the reaction is com- 
plete, from the formation of no further precipitate in 
the nearly clear liquid, as drops of silver nitrate mix 
with it. A chloride can be added to precipitate silver 
from a solution of its nitrate in the same manner, and 
so delicate is the reaction that a method based on it is 
still employed in many mints for determining the 
amount of silver in bullion, coin, or other alloy. 

For the determination of chlorides in solutions, es- 
pecially in urine, other methods, more convenient and 
fully as accurate, are employed. If a weak solution 
of silver nitrate be added to a neutral solution of a 
chloride containing enough potassium chromate to 
impart a slight yellow color to it the silver combines 
with the chlorine first and then, only after this has 
been completely precipitated, with the chromic acid 
to form brick-red silver chromate. In such a mixture 
the appearance of the first tinge of red is the indica- 
tion that the chlorine has been wholly precipitated. 
The neutral chromate is used here as the indicator. As 
each drop of silver nitrate solution falls into the solu- 
tion of chloride and chromate, a transitory reddish 
color may appear before the reaction is completed, but 
this vanishes on shaking or stirring the liquid and 
becomes permanent only when the chlorides are com- 
pletely combined with silver. In using this method 
the following solutions are employed : 

(a) Standard Silver Nitrate Solution, n/io. — Dissolve 
16.99 grams of pure fused silver nitrate in distilled 
water and dilute to 1 liter. Silver nitrate can 



THE DETERMINATION OF PHOSPHATES, ETC. 1 57 

usually be obtaiued of sufficient purity for the pur- 
pose, from dealers in fine chemicals, but should 
be fused in a porcelain crucible at a low tempera- 
ture before being weighed out. The correctness 
of the solution may be tested by the following : 

(b) Standard Sodium Chloride Solution, n/io. — Dissolve 
5.85 grams of pure, dry, recrystallized sodium 
chloride in distilled water and dilute to 1 liter. 

(V) Potassium Chromate Indicator. — Dissolve 10 grams 
of pure crystals, free from traces of chlorine, in 
100 cc. of distilled water. 

To test the accuracy of the standard silver solution 
fill a burette with the same and then measure into a 
beaker 25 cc. of the salt solution and add a few drops 
of the indicator. Now slowly run solution from the 
burette into the beaker, shaking the latter meanwhile, 
and continue until the curdy white precipitate shows 
a tinge of red from the presence of a little chromate 
formed. Exactly 25 cc. of the silver solution should 
be needed for this. One cc. of the silver solution so 
made precipitates 3.55 milligrams of chlorine or shows 
5.85 milligrams of sodium chloride. 

This method cannot be applied directly to the urine 
because of its yellow color which obscures the end re- 
action, and because, also, of the presence of certain 
organic matters which interfere to some extent. There- 
fore proceed as follows : Measure out accurately into a 
platinum or porcelain dish 10 cc. of the urine and add 
about 2 grams of potassium nitrate and 1 gram of dry 
sodium carbonate, both free from chlorides. Evapo- 
rate to drvness on the water-bath and then heat over 



158 URINE ANALYSIS 

the free flame, at first gently and finally to a higher 
temperature until the mass fuses. The organic mat- 
ter is destroyed by the nitrate, leaving finally a white 
molten residue. Allow it to cool, dissolve in water 
and add enough pure nitric acid to give a faint acid 
reaction. This destroys the carbonate. The slight 
excess of nitric acid must in turn be neutralized and 
this is done by adding a little precipitated and thor- 
oughly washed (chlorine-free) calcium carbonate. Pour 
the solution now into a flask, rinse the dish thoroughly, 
and add the rinsings to the liquid in the flask. Next 
add 2 drops of the chromate indicator and then the 
~ silver solution, until the faint red of silver chromate 
mixed with the chloride appears, showing the end of 
the titration. If the urine contains sugar or albumin 
in more than traces evaporate and heat with the so- 
dium carbonate first and then add the nitrate, a little 
at a time, to the charred mass to avoid too explosive 
an oxidation. The titration of the residue from the 
urine is therefore similar to the process by which the 
correctness of the silver solution was determined above. 
If in the titration 22 cc. of the silver solution were 
used we have 22 X 3.55 = 78.10 milligrams of chlo- 
rine in the 10 cc. of urine, corresponding to 128.7 
milligrams of sodium chloride. The sodium carbon- 
ate should not be omitted in this method as without 
it there is danger of loss of chlorine by volatilization. 
With care it gives excellent results but at present is 
not as generally employed as is the next one. 

Volhard's Method. — We have here a method by which 
the chlorine in urine can be quickly and accurately 



THE DETERMINATION OF PHOSPHATES, ETC. 1 59 

determined without fusion. The principle involved 
in the process is this. If to a chloride solution a defi- 
nite volume of standard silver solution be added, and 
this in excess of that necessary to precipitate the chlo- 
ride, the amount of this excess can be found by an- 
other reaction, subtracted and leave as the difference 
the volume actually needed for the chloride. The re- 
action for the excess depends on these facts. A thio- 
cyanate solution gives with silver nitrate solution a 
white precipitate of silver thiocyanate, AgSCN. It 
also gives with a ferric solution a deep red color 
due to the formation of soluble ferric thiocyanate, 
FeS (CN) . If the silver and ferric solutions are mixed 

3 y 3 

and the thiocyanate added the second reaction does 
not begin until the first is completed; that is, the sil- 
ver must be first thrown down as white thiocyanate 
before a permanent red shade of ferric thiocyanate ap- 
pears. The presence of silver chloride interferes but 
slightly with these reactions. Therefore, if we have 
a thiocyanate solution of definite strength we can use 
it with the ferric indicator to measure the excess of 
silver used after precipitating the chlorine of a solu- 
tion. 

The reaction between silver nitrate and a thiocyan- 
ate is expressed by the following equation : 

AgN0 3 + NH 4 SCN = AgSCN + NH 4 NO,. 

For 16.99 milligrams of the silver nitrate we use 
7.6 milligrams of the thiocyanate. In this method the 
standard solutions required are 

(a) Standard Silver Nitrate Solution, n/io. — Made as 
before with 16.99 grams of the fused salt to the 



l6o URINE ANALYSIS 

liter. As the solutions are used with nitric acid 
present, the standard can also be made by weigh- 
ing out accurately 10.79 grams of pure silver and 
dissolving it, in a flask, in pure nitric acid. Most 
of the excess of nitric acid is removed by evapo- 
ration, and air is blown through to drive out ni- 
-trous fumes. The solution is cooled and diluted 
to 1 liter. 

(b) Standard Thiocyanate Solution, n/io. — This may be 
made of the potassium or ammonium salt, but the 
latter is more commonly used. Weigh out about 
7.7 grams of the pure crystals, dissolve in water 
and make up to 1 liter. • Determine the exact 
strength as explained below. The true weight 
cannot be weighed out directly because the other- 
wise pure salt is frequently a little moist, and be- 
cause further the salt, pure to begin with, under- 
goes frequently a slight change on standing. 

(c) Ferric Solution as Indicator. — Use for this a nearly 
saturated solution of ammonium ferric sulphate 
(ferric alum) free from chlorine. 

To find the exact strength of the thiocyanate solu- 
tion proceed as follows: Measure into a flask or beaker 
25 cc. of the fjj silver solution and add to it 2 or 3 cc. 
of the ferric indicator. This gives some color and a 
slight opalescence. Now add about 2 cc. of pure 
strong nitric acid, which removes the color and clears 
the mixture. Into this, from a burette, let the thio- 
cyanate solution flow, a little at a time, shaking after 
each addition. A red color appears temporarily, but 
vanishes on shaking. After a time this red disappears 
more slowly, which shows that the end of the reaction 



THE DETERMINATION OF PHOSPHATES, ETC. l6l 

is near. The burette solution is therefore added more 
carefully, best by drops, until at last a single drop is 
sufficient to give a permanent reddish tinge. Some- 
thing less than 25 cc. should be used for this. Re- 
peat the test and if the same result is found dilute the 
thiocyanate solution so as to make 25 cc. of the vol- 
ume used in the titration. For instance, if 24.2 cc. 
were required 900 cc. of the solution may be diluted 
in this proportion : 

24.2 : 25 : : 900 : x .'. x = 929.8. 

We have now a standard thiocyanate solution corre- 
sponding exactly to the silver solution. To test it 
further and illustrate its use with chlorides measure out 
25 cc. of the -— sodium chloride solution described a 
few pages back, and add to it, from a burette, exactly 
30 cc. of the silver nitrate solution, then the ferric in- 
dicator and the nitric acid as given above. Shake the 
mixture and filter it through a small filter into a clean 
flask or beaker. Wash out the vessel in which the 
precipitate was made with about 20 cc. of pure w 7 ater, 
pouring the washings through the filter. Then wash 
the filter with about 20 cc. more of water allowing the 
washings to mix with the first filtrate. This mixed 
filtrate contains all the silver used in excess of the 
chloride. Now bring it under the thiocyanate burette 
and add this solution until a reddish tinge becomes 
permanent. Exactly 5 cc. should be necessary for 
this. 

The chlorides of the urine may be treated in about 
the same manner. To a measured volume of the urine, 
usually 10 cc, an excess of silver nitrate solutionis 
11 



1 62 URINE ANALYSIS 

added, 25 cc. with most urines is enough, and then 
the indicator and acid. The mixture is filtered, the 
precipitate washed, and in the filtrate the excess of 
silver is found by thiocyanate as above. But it occa- 
sionally happens that the addition of nitric acid to the 
urine develops a red color which obscures the end re- 
action. To avoid this the urine titration should always 
be made in the following manner : 

To 10 cc. of urine add 2 to 3 cc. of pure strong nitric 
acid, then the ferric indicator and three drops of a 
saturated, chlorine-free, solution of potassium perman- 
ganate. This gives at first a very deep red color, but 
it soon fades and with it the urine colors, by oxidation. 
It is not well to add more of the permanganate than 
here given. 

To the yellow solution add now 25 cc. of the stand- 
ard silver solution, shake well, and filter. Wash the 
beaker and filter thoroughly, as above described, and 
in the mixed filtrate and washings find the excess of 
silver. If in doing this the first drops of the thiocya- 
nate solution added produce a deep red color it shows 
that too little silver had been used in the first place. 
Make, therefore, a new test, using more silver nitrate 
solution, 30 to 50 cc. 

To illustrate, if we use for 10 cc. of urine, 25 cc. of 
silver nitrate, then 3.4 cc. of the thiocyanate, 25 — 3.4 
= 21.6, the amount of silver nitrate solution actually 
needed for the chlorides. 21.6 X 3.55 = 76.68 mil- 
ligrams of chlorine in the 10 cc. of urine, which cor- 
responds to 126.36 milligrams of NaCl in the 10 cc. 
or to 12.636 grains per liter. If the urine tested had 



THE DETERMINATION OF PHOSPHATES, ETC. 1 63 

a specific gravity of 1.020, this would equal 1.24 per 

cent. 

Sulphates 

Sulphur occurs in urine in several classes of com- 
pounds. It is most abundant in the ordinary sulphates, 
as sodium sulphate, potassium sulphate, and others, 
and is found also in the so-called ethereal sulphates or 
alcohol sulphates, of which the combination with 
phenol is a good illustration. These are all normal 
products, but the ethereal sulphates may be greatly 
increased pathologically. 

The total amount of sulphuric acid excreted daily, 
expressed as SO , amounts to about 2 grams; approxi- 
mately one-tenth may be considered as held in ethereal 
combinations, and the rest in the mineral sulphates. 
Many foods contain traces of sulphates and these traces 
are excreted as such ; but the larger part of the ex- 
creted sulphuric acid must be considered as formed 
from the sulphur in the proteids consumed as diet. 
Analyses show that all true proteids contain between 
1 and 2 per cent, of sulphur ; this in the body becomes 
oxidized to the sulphate form and is eliminated by the 
urine. Experiments on many individuals have shown 
that with a meat diet, in which proteids are, of course, 
relatively abundant, the amount of sulphuric acid ex- 
creted is increased, while with a diet of fruits and 
vegetables, with relatively low proteids, the acid is 
diminished. 

The amount of ethereal sulphuric acid found in the 
urine is a rough measure of the extent of putrefactive 
changes taking place in the body. Some of these pu- 



164 URINE ANALYSIS 

trefactive changes are normal in the intestines and are 
in constant operation ; the formation of urinary indi- 
can may be explained in this manner. Indol is pro- 
duced by putrefaction, and this oxidizes to indoxyl 
C 8 H 6 NOH. The latter, in turn, combines with sul- 
phuric acid to yield the ethereal sulphate, indican, 
C 8 H 6 NHS0 4 or C 8 H 6 NKS0 4 . Under pathological con- 
ditions, as in certain fevers, in peritonitis, in carcinoma 
of the stomach or intestines, the extent of putrefaction 
is greatly augmented, consequently there is an in- 
creased production of indol and the allied body, skatol. 
Possibly the larger part of these bodies is eliminated 
by the feces, but another portion is absorbed by the 
blood and oxidized. This oxidized part combines 
with sulphuric acid or acid sulphates in the blood to 
be subsequently excreted in the forms mentioned. 
Under such circumstances we find in the urine an in- 
creased amount of organic sulphates with a diminished 
amount of the mineral sulphates. Practically the same 
increase in ethereal sulphates is observed after the use 
of certain medicaments containing phenols or similar 
bodies. In cases of poisoning by common phenol 
there may be a nearly complete disappearance of the 
mineral sulphates from the urine. The consumption 
of certain foods and .condiments, rich in aromatics, is 
followed by a like change in the proportion of excreted 
ethereal sulphate. 

It will be recognized, therefore, that at times the 
determination of sulphates may have considerable 
clinical importance. Unfortunately, we have no 
method by which these determinations may be quickly 



THE DETERMINATION OF PHOSPHATES, ETC. 1 65 

made and with accuracy; but the qualitative deter- 
mination of an increase in the organic sulphates may 
be readily made after a little practice. The reaction 
depends on these principles : Barium chloride produces 
an immediate precipitate in a solution containing a 
sulphate, with or without the addition of acetic acid. 
In a solution of an ethereal sulphate made acid with 
acetic acid, barium chloride does not produce a pre- 
cipitate, even after moderate heating, but if hydro- 
chloric acid be now added and the mixture be warmed 
a precipitate will form and settle out. Therefore, if 
we have together two sulphates, for instance, potas- 
sium sulphate and phenyl-potassium sulphate, K 2 S0 
and C 6 H 5 KS0 4 , they may be separated by precipita- 
ting the sulphate of the K 2 S0 first, then that of the 
other after decomposing it by hot hydrochloric acid, 
which produces phenol and acid sulphate : 

C 6 H 5 KS0 4 -f H 2 = C 6 H 5 OH + KHS0 4 . 

Detection of Sulphates in Urine 

To show the presence of the sulphates in urine we 
may proceed in this way : To 50 or 100 cc. of urine, 
filtered, if necessary, add enough acetic acid to impart 
a good acid reaction. This will prevent the precipi- 
tation of phosphates and carbonates. Then add barium 
chloride in small amount and allow the mixture to 
stand for the separation of the precipitate of barium 
sulphate. Filter this off after a time and to the filtrate 
add a few cubic centimeters of pure dilute hydrochlo- 
ric acid and boil. If a new precipitate forms this 
points to the presence of the organic sulphates. 



1 66 URINE ANALYSIS 

If the urine contains oxalate a precipitate of barium 
oxalate may form with the first barium sulphate, but 
as the oxalate is soluble in hydrochloric acid the first 
precipitate may be treated with this acid to see whether 
it is soluble or not. If the urine contains albumin 
this must be separated by coagulating with a little 
acetic acid, warming, and filtering. The tests are then 
made on the filtrate. As the normal amount of organic 
sulphates in the 50 or 100 cc. of urine taken for a 
qualitative test is small, the precipitate will befarfrom 
heavy, but if the physician, by examination of a suffi- 
cient number of samples, once makes himself familiar 
with this normal appearance he will soon learn to de- 
tect an abnormally large amount without resorting to 
more complex methods. This skill can be acquired 
only by experiments on many samples of healthy urine. 
Determination of Sulphates 

The volumetric processes for the measurement of 
sulphates in urine are not convenient and, besides, are 
not extremely accurate. The usual gravimetric pro- 
cess, while accurate, is one which can be carried out 
only by a chemist in a properly equipped laboratory. 
It is therefore not clinically available and cannot be 
described at length in this place. It may be simply 
stated that the precipitates formed from a given vol- 
ume of urine, as in the qualitative test, are carefully 
collected on filters, thoroughly washed, dried, and 
weighed. From this weight of BaSO the acid may 
be calculated as SO or in any other form as desired. 
For the details of methods of gravimetric analysis 
special works on analytical chemistry must be consulted. 



Chapter VIII 

AMMONIA, XANTHIN AND ALLIED BODIES, 
ANDCREATININ 

Ammonia 

A small amount of ammonia is present in combina- 
tion in fresh normal urine, while in old urine the 
amount may be large from the decomposition of urea. 
With this we have nothing to do. The normal occur- 
rence of ammonia in urine may be credited in part to 
traces of ammoniacal salts taken with the food, but 
principally to the product formed by proteid disinte- 
gration. If the theory of the formation of urea from 
ammonium carbonate be correct, then the small amount 
of ammonia found in the urine, in excess of that taken 
directly with the food, could be referred to that left 
over by a failure to complete this reaction in the body : 
(NH 4 ) 2 C0 3 = CO(NH 2 ) 9 + 2H 2 0. 

It has been shown that ammonia in the urine is in- 
creased by a flesh diet, as is also urea, and that by a 
vegetable diet it is low. The average normal amount 
is about 0.7 to 0.8 gram daily, but the amount may be 
increased to 1.5 grams daily, or sink to 0.3 or 0.4 gram. 

Pathologically there seems to be a marked increase 
of excreted ammonia in fevers and the amount is 
usually found to vary with the greatly increased acid 
excretion. This has been noticed in diabetes mellitus 



1 68 TRINE ANALYSIS 

where a marked elimination of oxybntyric acid or dia- 
cetic acid is sometimes observed. The determination 
of excreted ammonia may, therefore, have practical 
value in diagnosis as bearing on the progress of the 
pathological condition. 

Determination of Ammonia 

As ammonia is always present in urine in some 
amount, qualitative tests have little value, and we pro- 
ceed immediately to quantitative methods. As the 
ammonia present is in combination as a salt it must 
be liberated by the action of a strong alkali, but in 
the choice of one for this purpose we are limited by 
the fact that the hydroxides of sodium and potassium 
have a decomposing action on urea and other nitro- 
genous bodies in urine and cannot, therefore, be used. 
Milk of lime is free from this objection and may be 
employed. The experiment is so arranged that the 
liberated ammonia may be absorbed by a measured 
volume of standard sulphuric acid, the amount of this 
neutralized being the measure of the ammonia ab- 
sorbed : 

H ; S0 4 - 2XH3 = (NH 4 ) 2 S0 4 . 

98 parts by weight of the acid correspond to 34 parts 
of ammonia, NH . 

The test is best made in this manner: Measure 25 
to 50 cc. of the fresh urine into a shallow glass dish 
with a flat bottom resting on a ground glass plate. A 
so-called crystallizing dish may be used with advan- 
tage. On this support a triangle of glass, and on this 
triangle place a second glass dish, preferably smaller 



AMMONIA, XANTHIN AND ALLIED BODIES, ETC. 1 69 

than the first. Measure into this top dish 10 ce. of 
normal sulphuric acid and then add to the urine 10 
to 20 cc. of good fresh milk of lime. Cover the whole 
with a bell-jar, as shown in the illustration, and allow 
to stand several days. In this time the milk of lime 
expels the ammonia from the urine. The rim of the 
bell-jar must be ground to fit the plate perfectly and 
in addition to this, should be rubbed with a little 
tallow. 




Fig. 7- 

As the ammonia cannot escape into the air it is ab- 
sorbed by the normal sulphuric acid, and after a time 
may be measured by titration. It is necessary to allow 
the apparatus to stand three or four days to insure 
complete absorption, as part of the liberated ammonia 
collects at first with a deposit of moisture on the inner 
walls of the bell-jar, and is given up from here rather 
slowly. When absorption is supposed to be complete 
the bell-jar is removed and the acid in the upper dish 



170 URINE ANALYSIS 

titrated with weak standard alkali, preferably fifth 
normal sodium hydroxide, after adding- methyl orange 
as indicator. The preparation of the standard sul- 
phuric acid and the standard alkali is given in the ap- 
pendix. Before titrating, it is well to rinse the sides 
of the bell-jar down with a little pure water and add 
the rinsings to the acid. 

In illustration of the calculations to be made in this 
test assume the following case: Take 50 cc. of urine 
and 10 cc. of the normal sulphuric acid. Each cubic 
centimeter of the acid contains 49 milligrams of actual 
H SO and will absorb and combine with 17 milli- 
grams of NH . Suppose now that at the end of the 
experiment, after adding methyl orange to the acid in 
the glass dish, we add from a burette 32.5 cc. of the 
fifth-normal sodium hydroxide solution to reach the 
point of neutrality (that is, to combine with the acid 
in excess of that neutralized by the ammonia), this 
amount of alkali is equivalent to 6.5 cc. of normal 
alkali and neutralizes, therefore, 6.5 cc. of the normal 
acid. 3.5 cc. of the acid must have been neutralized 
by ammonia. Now, as each cubic centimeter of acid 
will absorb and neutralize 17 milligrams of NH , it 
follows that the total amount of ammonia present in 
the 50 cc. of urine must have been 

3.5 X 17 = 59-5 milligrams, 
or 1. 19 grams to the liter. 

This test must always be made on fresh urine, as 
that which has stood contains an excess of ammonia 
from the bacterial fermentation of urea. When it is 
desired to save the day's urine for examination it is 



AMMONIA, XANTHIN AND ALLIED BODIES, ETC. 171 

necessary to add a little phenol to the collecting ves- 
sel, enough to make about 2 per cent, of this body 
present in the volume collected. 

The Xanthin Bases 

Normal urine contains a number of complex nitro- 
genous bodies which in composition, and to some ex- 
tent in chemical behavior, are closely allied to uric 
acid. Of these substances the following are known : 

Xanthin C 5 H 4 N 4 2 

Heteroxanthin C H 6 N 4 O 2 

Or methylxanthin C 6 H,N 4 O a CH 

f Paraxanthin C 7 H 6 N 4 2 

\ Or dimethylxanthin C 6 H,N 4 8 (CHJ 

Guanin C 5 H 5 N 5 0. 

Hypoxanthin (sarkin) C 5 H 4 N 4 0. 

Adenin C 5 H 5 N 5 

Carnin C ; H 8 N 4 3 

Uric acid is represented by the formula, C H N O . 

These bodies are all distinguished by their very 
slight solubility in water, by their ready solubility in 
mineral acids, with which they form crystalline com- 
binations of slight stability, and by precipitates which 
they give with solutions of many 'heavy metallic salts. 
Some of these precipitates are formed in extremely 
dilute solution. Under the tests for uric acid it was 
explained that in the precipitation of this acid by am- 
moniacal silver nitrate, Haycraft's method, the results 
are slightly inaccurate because of the coprecipitation 
of other bodies. The xanthin bases are the products 
having the greatest effect here, as they all combine 



172 URINE ANALYSIS 

with the silver or silver oxide of the alkaline solution. 

The amount of these bodies present in normal 
urine is small ; xanthin is the most abundant and 
of this the daily excretion is about 30 milligrams. For 
the detection of some of the bases it is necessary to 
operate on large quantities of urine. 

For the clinical separation of the xanthin bodies no 
convenient method is available. By operating on a 
large volume of urine they may be obtained in a sil- 
ver precipitate along with uric acid. This precipitate 
is thoroughly washed, mixed with water, and finally 
decomposed by hydrogen sulphide to separate the sil- 
ver; after filtering off the sulphide and concentrating 
the filtrate to a small volume, the uric acid separates 
from the other substances almost completely. On 
filtering again a solution is obtained from which the 
bases may be thrown down by several special methods. 
Instead of using ammoniacal silver solution as a pre- 
cipitant phosphotungstic acid along with hydrochloric 
acid may be employed. This gives a bulky precipi- 
tate containing the bases and uric acid. This pre- 
cipitate is washed with weak sulphuric acid until free 
from chlorine, drained and heated with a solution of 
barium hydroxide which forms an insoluble urate and 
brings the bases into solution. After filtering, the ex- 
cess of baryta may be separated from the filtrate by a 
little sulphuric acid. On again filtering a solution is 
obtained from which the bases may be thrown down 
by ammoniacal silver nitrate. For the identification 
of the individual bases in this precipitate the reader is 
referred to Neubauer and Vogel's " Urine Analysis." 



AMMONIA, XANTHIN A^KD ALLIED BODIES, ETC. I 73 

Creatinin 

This product, having the formula C H N O, occurs 
normally in urine and is excreted to the amount of 
1.5 to 2 grains daily. It is, therefore, more abundant 
than uric acid. As it is readily soluble in water and 
acids it escapes detection except when looked for by 
special reagents. In weak solutions it is precipitated 
by phosphotungstic acid, phosphomolybdic acid, pic- 
ric acid, and especially by solutions of several heavy 
metallic salts. The precipitate given with a neutral 
solution of zinc chloride is the most characteristic. It 
gives certain color reactions also. The following test 
may be applied to urine. If acetone is present it must 
be expelled by heat. To about 25 cc. of this urine 
add half a cubic centimeter of a dilute solution of so- 
dium nitroprusside made alkaline with caustic soda. 
With, this the urine gives a ruby-red color, fading to 
yellow. Then add acetic acid in slight excess and 
warm. A green color soon appears, deepening finally 
to blue. 

To obtain a characteristic precipitate with zinc chlo- 
ride some preliminary treatment is necessary, which 
may be applied in this manner. To about 250 cc. of 
urine add milk of lime to alkaline reaction, and then 
a few r cubic centimeters of a 10 per cent, solution of 
calcium chloride. This gives a bulky precipitate of 
phosphates and urates which is filtered off. The fil- 
trate is made neutral with -a small amount of acetic 
acid and evaporated to the consistence of a sirup on a 
water-bath. This sirup is treated with strong alcohol 
(98 per cent.) to dissolve the creatinin and leave salts 



174 URINE ANALYSIS 

in insoluble form. After standing six hours the mix- 
ture is filtered and to the nitrate a strong solution of 
pure chloride of zinc in alcohol is added. The mix- 
ture is stirred and allowed to stand two days, in which 
time a crystalline precipitate of the double salt settles 
out. This has the composition (C 4 H 7 N 3 0) 2 ZnCl. 
The yield of this precipitate is somewhat increased by 
adding a small amount of sodium acetate solution be- 
fore adding the zinc chloride. This insures precipi- 
tation from a solution free from stronger acids. The 
precipitate is to be washed on a filter with alcohol, 
and a portion may then be examined by the micro- 
scope. Rosettes or bundles of fine needles are usually 
obtained. 



Chapter IX 

THE SEDiriENT FROfl URINE 

Urine is frequently cloudy when passed and on 
standing deposits a sediment of the substances impart- 
ing the cloudiness. Other urines which may appear 
perfectly clear at first also throw down deposits after 
a time. This is always the case with urine allowed 
to stand long enough to undergo alkaline fermenta- 
tion, when a precipitate of phosphates forms. The 
deposit is frequently caused by a change of temperature. 
Warm voided urine holding an excess of urates may 
be perfectly clear, but becomes cloudy as its tempera- 
ture goes down with the formation of a light reddish 
sediment. This is a perfectly normal action, and in- 
deed most sediments may be considered in the same 
light. Urine containing a deposit is not necessarily 
pathological. 

There are conditions, however, in which the sedi- 
ment is an indication of abnormality, and its exami- 
nation becomes important clinically. Certain sediments 
are pathological because of their origin, others be- 
cause of their amount. For instance, blood and 
pus corpuscles, casts of the uriniferous tubules of 
the kidney and a few other forms are not found 
normally in urine, and their presence is of impor- 
tance, whether observed in large or small quan- 
tity. Sediments containing phosphates, uric acid and 



176 URINE ANALYSIS 

urates, calcium oxalate and other salts, are common 
enough and usually attract no attention, but if the 
amount of these deposits is very large there may be 
attached to them clinical significance and they deserve 
study. 

In the examination of a sediment it is necessary to 
allow the urine to stand long enough to deposit the 
important forms it may contain, which may require 
twenty-four hours or more. For the deposition of a 
sediment the urine should be left in a place with an 
even temperature, preferably not above 15 C. Alow 
temperature favors the precipitation of urates, while 
decomposition may begin if the temperature be allowed 
to go up. Some of the light organic forms have a 
specific gravity so little above that of the urine that 
they may remain a long time in suspension. It is im- 
portant, therefore, to allow plenty of time for these to 
settle. If the weather is warm and there is no good 
means at hand for keeping the temperature of the 
urine down until the examination can be made, or if 
for any reason this must be delayed for some days, it 
is well to add some preservative to the urine; i. e., 
something to prevent fermentation. Many substances 
have been suggested for this purpose, some of which 
are very objectionable inasmuch as they form precipi- 
tates which often obscure what is sought for. Chloro- 
form is the simplest and at the same time one of the 
best substances which can be added. 

To 100 cc. of the urine to be set aside for tests add 
three or four drops of chloroform and dissolve by sha- 
king. It is not well to add more than this as there is 



THE SEDIMENT FROM URINE 1 77 

danger of leaving minute droplets undissolved, and 
these are confusing in the subsequent examination. 
The chloroform may be applied in the form of aqueous 
solution. Add about 10 grams of chloroform to a liter 
of distilled water and shake thoroughly ; about three- 
fourths will dissolve at the ordinary temperature; 25 
cc. of this saturated solution may be added to 100 cc. 
of the urine to be examined, which is then allowed to 
stand as before. 

Recently, formaldehyde has come into use as a urine 
preservative and is applied as is the chloroform. It 
must be remembered that both of these substances are 
reducing agents, and therefore should not be used with 
urine to be tested for sugar. 

After the deposit has settled pour off the superna- 
tant liquid very carefully and by means of a small 
pipette with a coarse opening transfer one or two drops 
to a perfectly clean glass slide. Clean a cover glass 
with great care and by means of small brass forceps 
lower it on the drop of liquid in such a manner as to 
exclude air bubbles. This can be done by lowering 
it inclined to the slide, not parallel with it, so as to 
touch the liquid on one side first. In settling down, 
the cover now pushes the air in front of it and gives 
a field generally free from bubbles. The slide is then 
examined under a microscope with a magnifying 
power of 250 to 300 diameters. Either natural or ar- 
tificial light may be used, but it must not be very 
bright. A very common mistake in the examination 
of urinary sediments by the microscope is to employ 
so high a degree of illumination that the lighter and 



178 



URINE ANALYSIS 



nearly transparent bodies are completely overlooked. 

Recently, centrifugal machines have been introduced 
which may be employed to settle the urine. The 
latter for this purpose is placed in strong test-tubes 
which are caused to rotate so rapidly that all suspended 
matters are thrown to the bottom of the tubes, these 
beino; hung- by the neck in such a manner that in ro- 




8 bis. 



tation the bottom flies up and outward. Some of these 
rotating machines are operated by hand, others by 
water power or electricity. A very convenient form 
run by a small w r ater motor is now to be had from 
dealers. It is simply necessary to attach the motor 
by means of a rubber tube to a faucet delivering water 



THE SEDIMENT FROM URINE 1 79 

under ordinary city pressure to obtain all the power 
necessary. 

Where the current furnished for electric lighting by 
the incandescent system is available, centrifugal ma- 
chines operated by small electric motors are even more 
convenient. The cut to the right shows a small ma- 
chine which has given excellent results. The wires 
from the motor in the base of the machine are attached 
by a socket in place of the common incandescent lamps 
now everywhere used. The cut to the left shows a 
hand-power machine. 

A very high velocity is attained in these machines, 
by aid of which the deposit may be secured in min- 
utes instead of hours. The sediment is left in a very 
concentrated condition in the bottom of the tube, and 
from it the supernatant urine can be poured much 
better than when it precipitates in a beaker or bottle 
in the usual manner. 

Sediments from urine are commonly classed as or- 
ganized and unorganized, these divisions being then 
subdivided according to various plans. The impor- 
tant forms under each division are shown in the fol- 
lowing schemes : 

f Blood corpuscles. 

Mucus and pus corpuscles. 

Epithelium from various locations. 

Mucin bands, or threads. 
Sediments ^ Casts of the uriniferous tubules. 

Spermatozoa. 

Fragments of cancer tissue. 

Fungi. 
(^ Certain other parasites. 



l8o URINE ANALYSIS 

Uric acid. 
Various urates. 
Leucin and t\*rosin. 
Cy stiii. 
Cholesterin. 
Vn °l%tTl I Fat globules. 
Hippuric acid. 
Calcium carbonate. 
Calcium phosphate. 
Calcium oxalate. 
^ Magnesium phosphates. 

In addition to these there are often found in the 
urine certain bodies whose presence must be called 
accidental ; for instance, hairs, fibers of cotton, silk or 
wool, starch granules, bits of wood, mineral dust, etc. 
Some of these will be referred to later. 

Organized Sediments 

Blood Corpuscles 

Urine containing blood presents a characteristic ap- 
pearance easily recognized, unless it be present in very 
small quantity. If the reaction of the urine is acid 
the color is generally dark ; but if alkaline the shade 
is inclined to reddish. Blood corpuscles enter the 
urine from several different sources and their presence 
is usually a pathological indication, but not always, 
as they may come, for instance, from menstruation. 
The kidneys, or their pelves, the ureters, the bladder, 
the urethra, the vagina, or the uterus may be the seat 
of the lesion from which the blood starts, and its ap- 
pearance sometimes gives a clue to its origin. 



THE SEDIMENT FROM URINE l8l 

Fresh blood corpuscles are clear in outline and show 
distinctly their biconcavity. But corpuscles which 

O°o o 

Fig. 9. Human blood corpuscles, 400 diameters. 

have been long in contact with the urine become much 
swollen, less distinct in outline, often biconvex, or 
nearly spherical even, and lighter in color. As long 
as the reaction of the urine is acid the corpuscles re- 
main comparatively fresh in appearance but with the 
beginning of the alkaline reaction disintegration and 
loss of color soon set in. 

The microscopic recognition of blood in urine is 
easy enough if it is not too old. The fresh, red cor- 
puscles of human blood have a mean diameter of about 
0.0077 mm - 5 hut when swollen by absorption of 
water they are somewhat larger. When seen on edge 
they appear as shown at the left in the figure above. 
If presenting the flat side to the eye, they appear as 
disks whose centers grow alternately light and dark 
by changing the focus of the instrument. In old urine, 
especially with alkaline reaction, they appear as granu- 
lated spheres, shown at the left of the figure. In 
all cases the color is more or less yellowish. It is gen- 



I 82 URINE ANALYSIS 

erally assumed that the paler washed-out corpuscles 
come from lesions higher up, from the pelvis, or kid- 
ney even, while the brighter fresh blood suggests a 
lesion nearer the point of discharge ; that is, from the 
bladder or urethra. This is pretty certain to be the 
case if the blood is discharged but little mixed with 
the urine and settles rapidly as a distinct mass. 

Mucus and Pus Corpuscles 

These are white corpuscles somewhat larger than 
the red blood corpuscles and spherical in outline. The 
term leucocyte is frequently applied to these as well as 
to the so-called white corpuscles of blood. Their size 
varies greatly but the average diameter may be given 
as 0.009 mm - All these corpuscles present, when 
fresh, a slightly granular appearance and occasionally 
show one or more nuclei. The addition of a little 
acetic acid to the sediment brings the nucleus out dis- 
tinctly so that it ma}- be seen under the microscope as 
a characteristic appearance. 

Mucus corpuscles in small number are normally 
present in urine, but pus corpuscles enter the urine as 
a constituent of pus itself which is an albuminous prod- 
uct discharged from suppurating surfaces and not nor- 
mal. In a former chapter it was shown that the re- 
actions of mucus and albumin are distinct, but urine 
containing pus always affords reactions for albumin. 
Pus in urine tends to form a sediment at the bottom 
of the containing vessel, and may be recognized by 
the following method : 

Donne's Test. — Pour the urine from the sediment 



THE SEDIMENT FROM URINE 1 83 

and add to the latter an equal volume of thick potas- 
sium hydroxide solution, or a small piece of the solid 
potassa. Stir with a glass rod. The strong alkali 
converts the pus into a thick viscid mass closely resem- 
bling white of ^gg. Sometimes this is so thick that 
the test-tube containing it can be inverted without 
spilling it. In alkaline urine this glairy mass is some- 
times spontaneously formed. 






W*** &b 



•• 



fa 



Fig. 10. Pus corpuscles, 400 diameters. 

The appearance of the mucus or pus corpuscles in 
urine depends largely on the concentration of the lat- 
ter. In urine of low specific gravity the corpuscles 
absorb water and swell to larger size than normal, 
while in a highly concentrated urine they may give 
out water and become reduced in size and shrunken 
in appearance. 

To recognize them under the microscope transfer a 
few drops of the sediment to a slide, and cover as usual. 
If the nuclei are not distinct place a drop of diluted 
acetic acid on the slide at the edge of the cover glass. 
Part of the acid will flow under the cover and mix 
with the urine. As it does this the clearing up of the 



1 84 URINE ANALYSIS 

corpuscles, with appearance of the nuclei, can be very 
easily followed. Urine containing much pus is white 
and milky. The same appearance is often noticed 
with an excess of earthy phosphates, but the latter 
clear up with acids while the pus does not. 

Epithelium Cells 

Epithelium cells from different sources may appear 
normally in the urine, and the light cloud which sep- 
arates from normal urine on standing consists chiefly 
of these cells. When present in small amount this 
epithelium has usually no clinical importance, as it 
easily finds its way into the urine from the bladder, 
vagina, or urethra. An abundance of cells from these 




Fig. ii. Scaly and spherical epithelium. 

organs would, however, be considered pathological, 
pointing to a catarrhal condition. 

Unfortunately, it is not possible in all cases to de- 
termine the source of the cells, as found in urine, 



THE SEDIMENT FROM URINE 1 85 

partly because cells from different localities have fre- 
quently the same general appearance, and partly be- 
cause, owing to immersion in the urine, they become 
greatly changed from what they are in the tissue as 
shown by the microscopic study of sections. It is 
customary to make three rough divisions of the cells 
as found in the urine : 

1. Spherical cells. 2. Columnar or conical cells. 
3. Flat or scaly cells. 

The spherical cells are probably normally much 



*,.,;, I.J 



1 -* A 



^r-£$h 



(\ r. ' 



% m 



Fig. 12. Conical and spherical epithelium. 

flattened, but by absorption of water they become 
swollen and globular. These cells may be derived 
from several sources, as from the uriniferous tubules 
or from the deeper layers of the lining membrane of 
the pelvis of the kidney, or the bladder, or the male 
urethra. 

These cells have a well-defined nucleus resembling 
that of a pus cell. But they are much larger, and 



1 86 URINE ANALYSIS 

besides show the nucleus without addition of acid. In 
nephritis, or other structural diseases of the kidney, 
these round cells are found along with albumin, and 
their recognition is then a matter of importance as in- 
dicating a breaking down of the tubular walls. Some- 
times these cells form a variety of tube cast, to be de- 
scribed later. But it must be remembered that we 
cannot distinguish with certainty between the cells 
from the tubules and those from the other localities 
mentioned. 

Conical cells come generally from the pelvis of the 
kidney, from the ureters and urethra. Some of these 
cells are furnished with one or two processes, and are 
broad in the middle and taper toward each end, while 
the others are broad at the base and taper to a point. 

The large flat cells come from the vagina or blad- 
der, and it is generally impossible to distinguish be- 
tween them. Sometimes they are very nearly circu- 
lar, sometimes irregularly polygonal in outline. Some- 
times the vaginal epithelium is found in layers of 
scales, which appear thicker and tougher than the 
cells from the bladder, which occur singly. 

What was said about the decomposition of blood or 
pus cells in urine obtains also for the various epithe- 
lium cells. In acid urine they may maintain their 
distinct outlines many days, but in alkaline secretion 
they soon undergo disintegration, which makes their 
recognition practically impossible. In general the 
greatest importance attaches to the cells from the 
tubules of the kidney. The presence of albumin in 
more than minute traces in the urine would suggest 



THE SEDIMENT FROM URINE 1 87 

that any smaller spherical cells present may have had 
their origin in the kidney rather than in the bladder 
or male urethra. In general it may be said that urine 
containing large numbers of the smaller, round tubule 
cells with albumin will also show casts. 

Mucin Bands 

Urine containing much mucus sometimes exhibits 
a deposit consisting of long threads or bands, curved 
and bent in every direction. These bands are impor- 
tant because they are sometimes confounded with the 



Fig. 13. Mucin bands or threads. 

tube casts to be described next. They can be pro- 
duced in urine highly charged with mucus by the ad- 
dition of acids, and appear therefore sometimes spon- 
taneously when the urine becomes acid. These threads 
are sometimes covered with a fine deposit of granular 
urates and then bear some resemblance to granular 
casts. In general, however, they are relatively longer 
and narrower than the true casts of the uriniferous 



1 88 URINE ANALYSIS 

tubules. The mucin threads can occur, and frequently 
do occur, in urine entirely free from albumin, while 
true tube casts are usually associated with albumin, 
although not always, as will be explained below. The 
length and shape of the mucin threads may generally 
be relied upon to distinguish them from true casts. 

Casts 

The structures properly termed casts are seldom 
found in urine which does not contain albumin. They 
are formed in the uriniferous tubules, and, to a certain 
extent, are "casts" of portions of the same. Their 
specific gravity differs but little from that of the urine, 
for which reason they remain long in suspension. It 
is therefore necessary to allow the urine to stand some 
hours at rest, over night or longer, before attempting 
an examination, if a centrifuge is not at hand. 

Casts of the uriniferous tubules rarely appear in 
normal urine and their recognition is therefore a mat- 
ter of the highest importance in diagnosis. Much has 
been written on the subject of the origin of these bodies 
in the kidney and several theories have been advanced 
to account for their formation and chemical constitu- 
tion. Most of this discussion would be out of place 
in a work like the present dealing mainly with ques- 
tions of analysis, but enough will be given to aid the 
student in his examinations. It must be said that 
few subjects are more perplexing to the beginner than 
that of their certain recognition, because of the fact 
that some varieties are so transparent as to be almost 
invisible, while others are closely resembled by forma- 



THE SEDIMENT FROM URINE 1 89 

tions of entirely different nature, not pathological. 
With practice, however, these difficulties can be sur- 
mounted. 

Most of the bodies termed casts are formed of or- 
ganized structures or the remains of such, but another 
and rather common form consists of crystalline mat- 
ter, usually uric acid or fine granular urates. 



«KSF 6 



Fig. 14. Epithelium and blood casts; above, a bunch of 
urates or false casts. 

These bunches of urates have no pathological sig- 
nificance and are of frequent occurrence. Urine con- 
taining them clears up by heat, and the deposits them- 
selves are dissipated by weak alkali. While it is true 
that they resemble, to some degree, the so-called granu- 
lar casts referred to below, there are certain well-de- 
fined points of difference. The bunches of urates lack 
the coherence which can be observed in the true casts, 
and besides, the granulation is finer and more clearly 
defined. 



190 URINE ANALYSIS 

The fact that mucin bands occasionally appear cov- 
ered with a precipitate of granular urates has been re- 
ferred to. These aggregations are more compact than 
the loose bunches of urates just mentioned and much 






Fig. 15. Blood casts and granular casts. 

longer generally. Thev are also darker and there- 
fore more easily seen than are the casts proper or the 
urates. 

The true casts are made up of matter in which evi- 
dence of cell structure or transformation is visible. An 
acctirate classification of these bodies cannot yet be 
made, and, as said, authors differ regarding the im- 
portance of several forms and their origin. But for 
our purpose it will be sufficient to make the following 
rough division, which accords in the main with what 
is found in the text-books of urine analvsis : 



1. Blood casts. 

2. Epithelium casts. 

3. Granular casts. 



4. Fatty casts. 

5. Waxy casts. 

6. Hyaline casts. 



THE SEDIMENT FROM URINE 191 

What are termed blood easts consist of or contain 
coagulated blood, recognized by the corpuscles. Plugs 
of this coagulated matter are forced out from the tu- 
bules by pressure from behind, and form one of the 
most characteristic varieties of casts. They are gen- 
erally very dark in color, and easily distinguished from 
other matter. A representation of blood casts is given 
in the preceding cut. 

In epithelium casts the characteristic substance is 
the lining epithelium of the tubule. Sometimes this 
lining epithelium becomes detached in the form of a 
hollow cylinder, the walls consisting of the united 
cells. Again, the coagulated contents of the tubule in 
passing out may carry the. epithelium with it as a 
coating. In either case a grave disorder of the kid- 
neys is indicated, as acute nephritis, or other disease 
in which a profound alteration of the internal struc- 
ture of the organ is involved. 

What are termed granular casts, proper, appear in 
a variety of forms, produced probably by the disinte- 
gration of blood or epithelium casts. 

There is no uniformity in the fineness of the granu- 
lation ; sometimes a high amplification is necessary to 
disclose the structure. Occasionally blood corpuscles, 
epithelium, fat globules and crystals can be detected in 
them, and when derived from blood cast disintegration 
they usually have a yellowish red color, which makes 
their recognition comparatively easy. In outline they 
are generally regular, with rounded ends, one of which 
is somewhat pointed. Frequently, however, they ap- 
pear to be broken, the ends showing irregular fracture. 



192 URINE ANALYSTS 

Fatty casts contain oil drops produced by some va- 
riety of fatty degeneration of the tissues of the kidney. 
These oil drops may form coherent bunches, or they 
may be held by patches of epithelium. It also hap- 
pens that epithelium or granular casts may be partially 
covered by oil drops. The name, fatty cast, is applied 
to those in which the fat globules predominate. Along 
with these globules the microscope sometimes shows 
crystals of free fatty acids, and probably also of soaps 
containing calcium and magnesium. 

Waxy casts consist of the peculiar matter produced 







■ — - ■ ■ ■ "' 1 

Fig. 1 6. Waxy and hyaline casts. 

by amyloid degeneration of the kidney. They have 
a glistening wax-like or vitreous appearance, and re- 
fract light very strongly. Sometimes they reach a 
great length, and they frequently are found with blood 
corpuscles or oil drops on the surface. They have 
been detected in several renal disorders. Illustrations 



THE SEDIMENT FROM URINE 1 93 

True hyaline casts are nearly transparent and hard 
to see unless the illumination is very carefully man- 
aged. To detect them it is often necessary to add a 
few drops of a dilute solution of iodine in potassium 
iodide to the sediment. This imparts a slight color 
which renders them visible. 

The hyaline casts seem to be formed by the passage 
of homogeneous matter from the tubules, leaving the 
epithelium behind. A cast is rarely perfectly hyaline, 
as at least an occasional blood corpuscle, fat globule, 
or epithelium cell will usually be found attached to 
it. Waxy casts may be looked upon as a special form 
of hyaline casts. Very imperfect representations are 
given in the above cut. 

In general, it must be said that the representation 
of these casts on paper is a very difficult matter. Or- 
dinarily they are drawn and printed much too heavy 
and dark. 

Hyaline casts do not necessarily indicate kidney 
disease, although this is usually the case. They have 
been found in urine free from albumin and under cir- 
cumstances not connected with renal disorders. 

The preservation of sediments containing casts is 
unusually difficult because of the nature of the material 
to be preserved. In urine of the slightest alkalinity 
their disintegration soon begins, so that the outlines 
are rendered indistinct, often making identification 
impossible. 

For temporary preservation the addition of chloro- 
form renders as good service as anything else. Many 
other sediments can be permanently mounted and kept 
13 



194 URINE ANALYSIS 

for future comparison but with casts this can rarely 
be done. 

Beginners are apt to overlook casts in their first ex- 
aminations. It must be remembered that some of 
them are nearly transparent and unless brought into 
proper focus they may not be seen at all. At the out- 
set students usually employ too bright a light in look- 
ing for casts. While no specific directions can be 
given regarding the intensity of illumination best 
suited to the purpose, this may be said that the light 
commonly found necessary in studying ordinary his- 
tological slides is far too bright to use in the search 
for casts. 

Practice alone, first under the direction of the in- 
structor, will indicate what is proper here. 

Spermatozoa 

These minute bodies, as found in the semen of man, 
have a mean length of about 0.050 millimeter. Nearly 
one-tenth of this is in the head portion. When ob- 
served in recently discharged semen they have a char- 
acteristic spontaneous movement by which they are 
propelled forward rapidly. This motion is soon lost 
if the semen is diluted with water or similar liquid. 
Hence, as usually seen in urine, they are entirely mo- 
tionless. They are found abundantly in the urine of 
men after coitus or nocturnal emissions, and also in 
spermatorrhea, when their presence is continuous and 
characteristic. 

In the urine of women they are likewise found after 
connection, and their detection here has often interest 



THE SEDIMENT FROM URINE 195 

from the medico-legal standpoint. The proof of rape 
can often be established by the recognition of sperma- 
tozoa. 

Although their motion is soon lost in foreign liquids 
the substance itself of the spermatozoa is not readily 
destroyed. In this respect they are more permanent, 
probably, than all other organized structures found in 
the urine and can be readily distinguished after many 



^ v ^ ^H So W^o \w 



1 OsVfi^ *v \ 



^W;^ <>VV\ 



Fig. 17. Human spermatozoa. 

days or months even in urine, or in sediments which 
have become dry. 

For their recognition a power of 250 diameters is 
sufficient, but 350 to 400 diameters is preferable. With 
this higher amplification it is possible to readily dis- 
tinguish between spermatozoa and certain fungus 
growths bearing some resemblance to them. 

Cancer Tissue 

Fragments of cancerous and other morbid growths 
are occasionally met with in the urine, and when cer- 
tainly recognized are an important aid in diagnosis. 



196 URINE ANALYSIS 

The recognition of cancer cells along with the sev- 
eral kinds of epithelium cells found in nrine is diffi- 
cult and lies somewhat beyond the usual line of urine 
examinations. 




Fig. 18. Cancer cells which have been seen in urine. 

Sometimes the identification is comparatively sim- 
ple because of the unusual shape of the cells, or when 
they are present in great number, but this is not often 
the case. 

The cut shows some of these cells figured by Beale. 

Fungi 

The urine sometimes contains certain fungus growths, 
the recognition of which is important. These may 
have entered the urine after voiding, or they may have 
come from the bladder. 

Normal urine when passed is probably free from 
fungi of all kinds, but in a short time certain organ- 
isms enter it from the air or from other sources and 
become active in producing in it characteristic changes. 

The three important groups of fungi, the schizomy- 
cetes or bacteria, the hyphomycetes or molds and the 
blastomycetes or yeasts are represented in the organ- 



THE SEDIMENT FROM URINE 1 97 

isms sometimes found in the urine. The conditions 

under which they are found will be briefly explained. 

Of the bacteria the following have been observed : 

Micrococcus Ureae. — This is the exceedingly common 
form found on urine undergoing alkaline fermentation 
by which urea is converted into ammonium carbonate. 
It is usually introduced from the air and multiplies 
very rapidly under ordinary conditions. Nearly all 
old specimens of urine, unless containing some act- 
ive preservative, are found infected with this small 
organism. The micrococci are minute spherical bodies 
belonging to the suborder spherobacteria, and are 
found separate or in chains. They are the smallest 
of the organized forms occurring in urine and appear 
under a power of 250 diameters but little more than 
points. 

While generally finding their way into urine 
after it has been voided they are occasionally present 
in the bladder. It is usually held that under such 
circumstances they have been introduced by a dirty 
catheter or sound, although cases are on record where 
this has not been proved. 

In the bladder they give rise to alkaline fermenta- 
tion, so that the voided urine may show ammonium 
carbonate directly. 

Streptococcus Pyogenes is a pathogenic form some- 
times found in the urine in cases of infectious diseases. 

Sarcinae. — The genus sarcina is frequently classed 
with the spherobacteria and several species have been 
found in urine. The cells are larger than those of 



198 URINE ANALYSIS 

micrococcus ureae, and are arranged in groups of two 
or four usually. They are not pathogenic. 

Bacilli. — Several species of the genus bacillus axe. 
found in urine in disease. The most important of 
these are the typhoid bacillus, bacillus typhi abdomi- 
nalis,the tubercle bacillus, bacillus tuberculosis, and 
the bacillus of glanders, bacillus mallei These bacilli 
occur in urine only during the progress of the corre- 
sponding diseases and their detection is of the highest 
interest. A description of the methods to be followed 



m 
v> 


x 


'pod 


<*\ 


* 




« 


9 fl 




Fig. 19. Micrococci and other bacteria. 

for the certain demonstration of these bodies is not 
within the scope of this book, but must be looked for 
in the laboratory manuals of bacteriology. 

Spirilla. — Certain species of the genus spirillum 
have been found in urine. The best known of these 
is the spirillum of relapsing fever, spirillum Obermeiri. 
This is only found rarely and as its habitat is the 
blood of relapsing fever patients it must enter the 
urine through a hemorrhage into the kidney. Its 
form is that of a long, wavy spiral, which makes its 
detection somewhat easy. 



THE SEDIMENT FROM URINE 



199 



Although not pathogenic it is well to call attention 
to certain molds which may sometimes be seen in 
urine. The common blue-green mold, penicillium 
glaucum, is the best known of these, and is occasion- 
ally found in urine along with yeast cells. Another 
mold which has been found in urine is the oidinm 
lactis, commonly occurring in milk and butter. It has 
been observed in fermenting diabetic urine. Both of 
these fungi enter the urine after voiding. In urine 
which has stood sometime in a cool place the penicil- 
lium glaucum sometimes becomes covered with an in- 
crustation of urates or minute crystals of uric acid. 

Finally we have yeast cells in urine and sometimes 
in great numbers. Like other fungi they enter the 







Fig. 20. Yeast cells and common mold. 



urine from the air and when not very abundant have 
no significance. In great numbers the yeast cells sug- 
gest presence of sugar. The ordinary yeast plant, 
saccharomyces cerevisice, is shown, isolated and bud- 
ding, in the accompanying figure. 



200 URINE ANALYSIS 

Other Parasites 

Besides fungi the urine in rare cases contains other 
parasites of animal as well as vegetable nature. Some 
of these enter it accidentally from the air and have no 
interest, but a few are present in the urine when 
passed, and are of importance. Among these are cer- 
tain thread worms, the eggs of worms and hooklets of 
a species of tapeworm, the taenia echinococcns. 

These forms can appear in the urine only through 
rupture from some other organ, and while rare here, 
are common enough in Egypt and other tropical coun- 
tries. Urine containing eggs of worms, the worms 
themselves or fragments, usually contains blood or 
other evidence of rupture. 



Chapter X 

UNORGANIZED SEDIMENTS AND CALCULI 
Unorganized Sediments 

Uric Acid 

Among the more common of the unorganized sedi- 
ments found in urine this must be mentioned first. 
As was explained in the last chapter uric acid occurs 
normally in combination in all human urine. 

Some time after its passage urine often undergoes 
what has been spoken of as the acid fermentation by 
which a precipitate of urates and even free uric acid 
may appear. This reaction is in no sense due to a 
ferment process in the ordinary sense of the term, but 
is probably brought about by a purely chemical double 
decomposition. Urine contains acid sodium phos- 
phate and neutral sodium urate and it has been sug- 
gested that these react on each other according to the 
following equation : 
Na 2 C 5 H 2 N 4 3 + NaH 2 P0 4 = NaC B H,N 4 0, + Na 2 HP0 4 . 

The precipitate of acid urate settles out and forms 
a light reddish deposit. If the amount of acid phos- 
phates present is excessive the reaction may go still 
further, resulting in the precipitation of free uric acid. 
The well-characterized crystals of uric acid are often 
found with the sediment of fine urates. Sometimes 
this liberation and precipitation of the acid takes place 



202 



URINE ANALYSIS 



in the bladder, and the urine, as passed, shows the crys- 
tals or "gravel." If they are relatively large, which 




Fig. 21. Uric acid. 

is sometimes the case, their passage through the 
urethra may cause severe pain. 

As the illustrations show, uric acid occurs in a great 
variety of forms. The rosettes and whetstone-shaped 




Fig. 22. Uric acid. 

crystals are probably the most common, while long 
spicnlated forms are frequently seen. Pure uric acid 



UNORGANIZED SEDIMENTS AND CALCULI 203 

is colorless but as deposited from urine it is always 
reddish yellow, because of its property of carrying 
down coloring-matters. The crystals are often so 
large that their general form can be seen by the naked 
eye ; usually, however, they are minute. 

Uric acid crystals when once deposited are not read- 
ily redissolved by heat, but they go into solution by 
the addition of alkali. If the urine contains extrane- 
ous matter, as specks of dust, bits of hair, cotton or 
wool fibers, the crystals are very apt to deposit on 
them. 

Urates 

The common fine sediments of urine are usually 









o,® ^jtm^fflr •*.« 







Fig. 23. Common crystalline and granular urates. 

urates or amorphous phosphates. They can be most 
readily distinguished by their behavior with acids and 
on application of heat. Urates disappear on warming 
the urine containing them, while a phosphate sedi- 
ment is rendered more abundant. A urate sediment 
is little changed by acids, while the phosphates dis- 



204 URINE ANALYSIS 

solve completely if the urine is made acid in reaction 
with hydrochloric or nitric acid. The acid urates of 
sodium and ammonium are the most abundant and are 
shown in the cut. Acid ammonium urate may exist 
in urine which has become alkaline from the decom- 
position of urea and formation of ammonium carbon- 
ate, and may therefore be seen in company with the 
phosphate sediments. The other urates dissolve in 
alkaline urine. Like uric acid the urates appear in a 
great variety of forms, and there is still some uncer- 
tainty about the composition of some of their crystals 
which have been found in urine. 

Leucin and Tyrosin 

These two substances are of rare occurrence in urine 
and appear only under pathological conditions. Urine 
containing them shows usually strong indications of 
the presence of biliary matters as they generally are 
found in consequence of some grave disorder of the 
liver in which destruction of its tissue is involved. 
They have been most frequently found, and associated, 
in acute yellow atrophy of the liver and in severe 
cases of phosphorus poisoning. In general they must 
be considered as products of disintegration and are 
produced in the intestine in large quantity by bacte- 
rial agency in the last stages of the digestion of albu- 
minoids, as was pointed out in an earlier chapter. 

As both bodies are slightly soluble they may not be 
seen directly, but only after partial concentration of 
the urine. In pure condition leucin crystallizes in 
thin plates but from urine it separates in spherical 



UNORGANIZED SEDIMENTS AND CALCULI 



205 



bunches made up of fine plates or needles. These 
bunches are sometimes so compact that it is hard to 
distinguish between them and other substances, par- 
ticularly lime soaps and oil drops. Chemical tests 
must therefore be applied. If mercurous nitrate is 
added to a leucin solution and the mixture is warmed, 
metallic mercury precipitates. This test can be car- 




Fig. 24. Leucin spheres, tyrosin needles and cystin plates. 

ried out only when the substance is abundant enough 
to be purified by crystallization from hot water. Pure 
leucin, when strongly heated with nitric acid on plati- 
num, forms a colorless residue, which when heated 
with potassium hydroxide leaves an oil-like drop that 
does not wet the platinum. 

Tyrosin is usually seen in long needles, which some- 
times are bunched in the form of sheaves, and is more 
readily recognized than is leucin. Tyrosin heated 
with nitric acid on platinum turns orange-yellow, and 
leaves a dark residue which becomes reddish yellow 



206 URINE ANALYSIS 

by addition of caustic alkali. Solutions containing 
tyrosin, when treated hot with mercuric nitrate and 
potassium nitrite, turn red and finally throw down a 
red precipitate. 

Leucin and tyrosin may be present in the urine yet 
not show as a sediment. For their detection under 
these circumstances precipitate the urine with basic 
lead acetate and filter. Separate the excess of lead 
from the nitrate by hydrogen sulphide and filter again. 
Concentrate the filtrate on a water-bath to a sirup and 
treat it with a little absolute alcohol to remove urea. 
Some leucin may dissolve with the urea in this treat- 
ment. Next boil the residue with 60 or 70 per cent, 
alcohol, filter, concentrate the filtrate to a small vol- 
ume and allow it to stand in a cool place for crystalli- 
zation. If crystals appear their form indicates" whether 
they consist of pure tyrosin or a mixture of tyrosin 
and leucin. The latter, being more soluble in strong 
alcohol, can be separated by washing with this liquid. 
The final tyrosin residue can be used for the tests 
given above. 

The alcoholic solutions may contain leucin, which 
can be recognized after evaporation. Both leucin and 
tyrosin decompose readily in urine undergoing putre- 
factive changes ; it is therefore necessary to apply the 
test to urine as fresh as possible. 

Cystin 

This is a rare sediment, although it is found con- 
stantly in the urine of certain individuals. It crys- 
tallizes in thin hexagonal plates, small ones sometimes 



UNORGANIZED SEDIMENTS AND CALCULI 



207 



resting upon or overlapping large ones. The crystals 
are regular in form but variable in size and readily 
recognized. A rare form of uric acid crystallizes in a 
somewhat similar manner but the two substances differ 
in their behavior toward ammonia. To distinguish 
between them in the microscopic test place a drop of 
ammonia water on the slide and allow it to pass under 
the cover glass. Cystin dissolves, but, unless heated, 
uric acid does not. When the ammonia evaporates 
cystin reprecipitates. 

Cystin is precipitated from urine by addition of 
acetic acid. Mucin and uric acid may come down at 




Fig. 25. Cholesterin plates and fat globules. 

the same time. The precipitate is collected on a filter, 
washed with water and finally dissolved in ammonia. 
By neutralizing the ammoniacal filtrate with acetic 
acid and concentrating a little, it comes down in the 
characteristic form suitable for microscopic recognition. 

Cholesterin 
This substance occurs occasionally in urine, and 
has been found in cases of cystitis and chyluria. It is 



208 



URINE ANALYSIS 



recognized by its characteristic crystalline form, large, 
thin plates, shown in the cut (Fig. 25). These plates 
are nearly transparent but from their size cannot be 
overlooked. Cholesterin is a common constituent of 
biliary calculi. 

Fat Globules 

These are often seen in urine, but in most cases have 
not been voided with it. The}- can come from several 
extraneous sources, as from a catheter, from vessels in 
which the urine is collected or sent for examination, 




Fig. 26. Hippuric acid. 

from admixed sputum, etc., which facts should be 
borne in mind. 

Fat has been found in cases of fatty degeneration of 
the kidney and more abundantly in chyluria where 
communication seems to be formed between the lym- 
phatics and the urinary tract by the invasion of the 
small thread worms referred to above. 



UNORGANIZED SEDIMENTS AND CALCULI 209 

Hippuric Acid 
This acid is found normally in human urine in 
small amount. It may be found in large quantity 
after taking benzoic acid and may even appear in crys- 
talline form in the sediment. It has no pathological 
importance, ordinarily. 

Calcium Carbonate 
This is sometimes observed as a coarse, granular 
sediment which dissolves with effervescence in acetic 
acid. It occasionally forms dumb-bell crystals, and is 
devoid of pathological importance. 

Calcium Sulphate 

Crystals of this substance are rarely found in urine. 
They form long, colorless needles, or narrow, thin 
plates. 

Calcium Oxalate 

We have here one of the commoner of the crystal- 
line bodies observed in urine. 

This may be found in neutral or alkaline urine, but 
more commonly in that of acid reaction. It occurs 
normally and sometimes is very abundant, especially 
after the consumption of vegetables containing oxalic 
acid. 

Two principal forms of the crystals are found, the 
octahedral and dumb-bell crystals. 

The octahedra have one very short axis which gives 

the crystals a flat appearance. When seen with the 

short axis perpendicular to the plane of the cover 

glass, which is the common position, they appear as 

14 



2IO URINE ANALYSIS 

squares crossed by two bright lines. Sometimes they 
are seen on edge, and then present a rhomb in section 
with one diameter very much shorter than the other. 

A form of triple phosphate bears a slight resem- 
blance to calcium oxalate, but it is soluble in acetic 
acid, while the oxalate is not. 

The dumb-bells are much less common than the 
octahedra, and are found in several modified forms, as 
shown in one of the figures. 

The clinical significance of the oxalate is not clearly 





Fig. 27. Calcium oxalate. 

understood. It does not seem to be characteristic of 
any disease even when occurring in quantity. It has 
been found considerably increased in dyspeptic con- 
ditions, but not always, and many of the statements 
found concerning its significance seem to have been 
based on insufficient observations. 

Urine may contain a large amount of oxalic acid 
which does not show as a sediment, but must be found 



UNORGANIZED SEDIMENTS AND CALCULI 



211 



by precipitation with calcium chloride in presence of 
ammonium hydroxide. Acetic acid is then added in 
very slight excess and the mixture is allowed to stand 
for precipitation. 

The constant or prolonged excretion of large amounts 
of oxalic acid is spoken of as oxaluria. 

The Phosphates 

It was explained in Chapter VII that phosphates 

of alkali and alkali-earth metals occur normally in the 

urine, and a method was given for their estimation. 

As sediment we know several forms of calcium and 




Fig. 28. Triple phosphate. 

magnesium phosphates and the microscopic detection 
of these will be here explained. In normal fresh urine 
of acid reaction these phosphates are held in solution, 
but if the urine as passed is alkaline it is often turbid 
from the presence of basic phosphates held in suspen- 
sion. Urine which has stood long enough to undergo 
the alkaline fermentation always contains phosphates 



212 



URINE ANALYSIS 



in the sediment. Finally, it must be remembered that 
a neutral or very slightly acid urine, containing am- 
monium salts in abundance, may also deposit a crys- 
talline precipitate of ammonium magnesium phos- 
phate. The common phosphate sediments are those 
consisting of ammonium magnesium phosphate (triple 
phosphate), normal magnesium phosphate, neutral cal- 







Fig. 29. Neutral calcium phosphate and amorphous phosphate. 



cium phosphate, and mixed amorphous phosphates of 
calcium and magnesium. 

Triple Phosphate. — Of the crystalline phosphate de- 
posits this is the most abundant and at the same time 
the most characteristic. 

The crystals are the largest found in urine, and from 
their shape are sometimes spoken of as coffin-lid crys- 
tals. Ordinarily they are not found in perfectly fresh 
urine, but after it has undergone the alkaline fermen- 
tation they are generally present in profusion. 

Normal Magnesium Phosphate — Crystals having the 
composition, Mg (PO ) 2 .2 2H^O, are sometimes found 
in urine of nearly neutral reaction. They consist of 



UNORGANIZED SEDIMENTS AND CALCULI 213 

thin, transparent, rhombic plates with angles of approxi- 
mately 6o° and 120 . If urine containing this sedi- 
ment becomes alkaline, triple phosphate forms. 

Neutral Calcium Phosphate. — This has the composi- 
tion, CaHPO .2H 2 0, and is found in urine of neutral 
or slightly acid reaction. It crystallizes frequently in 
rosettes formed of wedge-shaped, single crystals, uni- 
ting at their apices. The cut shows some variations 
in the form. 

Amorphous Phosphates. — Finally we have the very 
common, finely granular, earthy phosphates in amor- 
phous condition. This sediment dissolves readily in 
weak acetic acid and is colorless. The common amor- 
phous urate sediment is colored and does not dissolve 
in acetic acid. On addition of sodium carbonate or 
hydroxide to urine, the precipitate which forms con- 
sists mainly of this phosphate. 

These several phosphates can be produced artificially 
and should be made for study and comparison. The 
normal magnesium phosphate can be made by dis- 
solving 15 grams of crystallized common sodium 
phosphate in 200 cc. of water and mixing this with 
3.7 grams of crystallized magnesium sulphate in 2000 
cc. of water. Enough sodium bicarbonate is added to 
give an amphoteric reaction and then the mixture is 
allowed to stand a day or more for precipitation. 

Crystals of triple phosphate of peculiar form are 
often obtained by adding ammonia to urine, and some- 
times a trace of ammonia is sufficient to throw down 
the crystals of neutral calcium phosphate. The latter 



2I 4 



URINE ANALYSIS 



can also be obtained by adding to a weak solution of 
crystallized sodium phosphate a trace of acid and then 
a very little calcium chloride solution. 

Foreign Matters 

The sediment of urine often contains foreign sub- 
stances which have become mixed with it accidentally. 
The most common of these are hairs, woolen, cotton 
or silk fibers, granules of starch, fat globules, dust and 




Fig. 30. L, linen fibers ; H, hemp fibers ; J, jute fibers ; 

B, cotton fibers; S, silk fibers; A, alpaca fibers ; 

E, fine wool ; W, common wool. 

sand granules, bits of woody fiber and remains of arti- 
cles of food. Some of these are represented in Figs. 
30 and 31. 

Urinary Calculi 

Calculi, like the sediments just described, are formed 
by the precipitation of certain substances from the 
urine, but in compact form. Occasionally a calculus 



UNORGANIZED SEDIMENTS AND CALCULI 



215 



consists of a single substance, as calcium oxalate or 
cystin, but in the great majority of cases a mixture of 
bodies is present, these being deposited usually in 
layers around a nucleus which serves as the founda- 
tion of the concretion. Calculi are built up much as 
certain forms of crystals are by successive depositions 
on a nucleus. Uric acid is a very common nucleus 
on which may be deposited urates, phosphates, organic 
matters, etc. 

Calculi are sometimes distinguished as primary or 
secondary. Primary calculi may be traced to an alter- 






Fig. 31. Left, pubic hair with spermatozoa ; center, hair of 
woman's head ; right, cat's fur. 

ation of the urine of such a nature that its reaction is 
constantly acid. The foundation for the concretions 
in this case is found in the kidney and they are built 
up of such substances as most easily deposit from acid 
urine. Secondary calculi are generally formed in the 
bladder, and have for nuclei matters precipitated from 
alkaline urine, as coagulated blood or other organic sub- 
stances. Sometimes fragments introduced into the 
bladder from without serve as the foundation for these 



2l6 URINE ANALYSIS 

secondary formations. Bits of catheters, remains of 
bougies, and other things have been found as the 
nuclei around which concretions have formed. The 
recognition of the nucleus is a matter of the first im- 
portance as this gives a clew to the determining cause 
active in the formation of the calculus. 

In making an examination, then, of a calculus, it is 
first cut in two by means of a very sharp thin saw. 
This exposes the nucleus which may often be recog- 
nized by the eye alone. If one of the halves be pol- 
ished it is often possible to discern distinctly the vari- 
ous layers grouped around the center. 

In a large number of cases examined by Ultzmann 
about 80 per cent, were found to contain uric acid as 
the nucleus. 

Chemical Examination 

In the chemical examination of a calculus several 
methods may be employed. We may begin by apply- 
ing certain preliminary tests designed to show the 
general nature of the stone. 

Heat Test. — Reduce some of the calculus to a pow- 
der and heat to bright redness on platinum foil. Two 
cases may arise : (a), the powder is completely con- 
sumed ; (/;), the pow T der is only partially consumed or 
not at all. 

Case (a). If this is the result of the incineration 
the following substances may be suspected : 

Uric Acid, which may be recognized by dissolving a 
little of the powder in weak alkali, precipitating by 



UNORGANIZED SEDIMENTS AND CALCULI 217 

hydrochloric acid and examining the precipitate by 
the microscope. 

Ammonium Urate. — This gives the above reaction 
under the microscope, and is further recognized by 
the liberation of ammonia when heated with a little 
pure sodium hydroxide solution. 

Cystin. — Dissolve some of the powder in ammonia, 
filter if necessary and allow drops of the filtrate to 
evaporate spontaneously on a slide. Cystin is then 
recognized by the microscope as already explained. 
Cystin contains sulphur which, on burning on the plat- 
inum foil, gives rise to a disagreeable sharp odor. If 
a little of the powder be heated with a mixture of po- 
tassium nitrate and sodium carbonate the sulphur is 
oxidized to sulphate, which may be recognized by the 
usual tests. 

Xanthin. — This is a rare substance in calculi. Those 
consisting wholly of xanthin are brown in color and 
take a wax-like polish. A method of recognition will 
be given below. 

Organized Matter. — Parts of blood cells, epithelium, 
precipitated mucin, pus corpuscles and similar sub- 
stances may become entangled with the growing stone 
and even form a large part of it. On burning, these 
bodies are recognized by the characteristic odor of 
nitrogenous matter. 

Case (b). When an incombustible residue is left 
on the platinum foil the stone may contain the follow- 
ing constituents : 

Calcium Oxalate. — Stones of this substance are very 



218 URINE ANALYSIS 

hard and break with a crystalline fracture. They are 
often called "mulberry calculi." When the powder 
is heated it decomposes, leaving carbonate, which may 
be recognized by its effervescence with acids. 

Calcium and Magnesium Phosphates. — They leave a 
residue in which the metals and phosphoric acid may 
be detected by simple tests of qualitative analysis. 
The ignited powder is soluble in hydrochloric acid 
without effervescence. When ammonia is added to 
this solution in quantity sufficient to give an alkaline 
reaction, a precipitate of triple phosphate or calcium 
phosphate appears, which may be recognized by the 
microscope. 

The above tests are generally sufficient to tell all 
that is practically necessary about the calculus. If 
more detailed information is desired a systematic 
analysis may be made according to the following 
scheme : 

Systematic Analysis — i. Reduce the calculus to a 
fine powder and pour over it some water and finally 
dilute hydrochloric acid in a beaker. W T arm gently 
half an hour, or longer, on the water-bath. Then 
allow to cool and filter. 

2. Treatment of the residue. It seldom happens 
that the calculus is completely soluble in the weak 
acid. A residue usually remains which may contain 
uric acid, xanthin, calcium sulphate, and remains of 
organized matter. To prove the xanthin treat the 
residue with warm dilute ammonia and filter. The 
filtrate contains the xanthin if it is present. Acidify 



UNORGANIZED SEDIMENTS AND CAECUEI 219 

it with nitric acid and add a small amount of silver 
nitrate solution. This produces a flocculent precipi- 
tate which dissolves by warming, and crystallizes on 
cooling in bunches of fine needles. 

In the residue free from the xanthin look for cal- 
cium sulphate by extracting with water and applying 
the usual tests. This solution may contain uric acid 
which is recognized by evaporation and crystallization 
after adding a little hydrochloric acid. In the final 
residue some uric acid may be also present. Dissolve 
in alkali, reprecipitate with hydrochloric acid, and 
examine any crystals which may form under the mi- 
croscope. 

3. Treatment of the hydrochloric acid solution. 
This may contain calcium oxalate, cystin, the phos- 
phates, and possibly some xanthin. Look for the last 
in a small portion of the solution. Make this portion 
alkaline with ammonia, add a few drops of calcium 
chloride solution, filter if a precipitate forms, and treat 
the filtrate with ammoniacal silver nitrate solution. 
In presence of xanthin a flocculent precipitate forms. 

Dilute the remaining and larger portion of the hy- 
drochloric acid solution with twice its volume of water, 
add enough ammonia to give a strong alkaline reac- 
tion, and then acetic acid to restore a weak acid reac- 
tion. By this treatment phosphates are held in solution 
while calcium oxalate, if present, precipitates. There- 
fore allow the mixture to stand half an hour and then 
filter off any precipitate which appears. This precipi- 
tate may contain cystin as well as calcium oxalate. 
Cystin may be dissolved by pouring ammonia on the 



220 URINE ANALYSIS 

filter, and on evaporating the ammoniaeal solution is 
obtained in form suitable for microscopic examination. 

The residue free from cystin is dried and heated to 
redness on platinum foil. This treatment converts 
calcium oxalate into carbonate. Place the foil in a 
beaker and add some dilute acetic acid ; an efferves- 
cence shows the carbonate. To the clear solution add 
next some ammonium oxalate which gives a white 
precipitate of calcium oxalate, if the latter metal is 
present. 

We have next to look for the phosphates and bases 
in the acetic acid solution obtained after filtering off 
cystin and calcium oxalate. More calcium may be 
present, in excess of that combined as oxalate, which 
may be recognized by adding a little solution of am- 
monium oxalate. If a precipitate forms treat the 
whole of the liquid with the ammonium oxalate; after 
warming on the water-bath, allow to stand an hour 
and filter. Concentrate the filtrate in platinum to a 
small volume, transfer to a large test-tube and add 
enough ammonia to produce an alkaline reaction. If 
a precipitate appears now it must consist of magnesium 
phosphate, showing both magnesium and phosphoric 
acid as present in the original. If no precipitate ap- 
pears magnesium is absent but phosphoric acid may 
still be present. To find it divide the ammoniaeal 
liquid into two portions. To one add a few drops of 
magnesia mixture, and to the other add nitric acid in 
slight excess and then ammonium molybdate reagent. 
Both tests should yield the reactions characteristic of 
phosphates, if present. 



UNORGANIZED SEDIMENTS AND CALCULI 22 1 

This procedure serves for the recognition of the im- 
portant constituents of calculi. But ammonium, po- 
tassium, and sodium compounds are sometimes present 
and may be recognized readily. To detect ammonium 
salts the original calculus powder may be heated with 
pure potassium hydroxide solution, or the hydrochloric 
acid solution of the calculus may be neutralized and 
heated with the same solution. The ammonia is rec- 
ognized by the odor or by its reaction on moistened 
red litmus paper. 

To recognize the alkali metals a solution of the 
powdered calculus in hydrochloric acid is treated with 
pure ammonia and a little ammonium carbonate in 
excess. The precipitate formed is allowed to settle 
and filtered off. The filtrate is then evaporated to dry- 
ness in a platinum dish and the residue strongly 
heated to drive off all ammonium salts. What is now 
left contains sodium and potassium if they were pres- 
ent in the original. Moisten this final residue with 
water and a drop of hydrochloric acid and test with 
a platinum wire in the flame of a Bunsen burner, 
using a deep blue glass when looking for potassium. 
Only a very intense yellow color can be taken as in- 
dicative of sodium. 



APPENDIX 



Tables and Notes 



Tables of Weights and fleasures 

The Metric System 
ijineter = ioo centimeters, cm. = iooo millimeters, mm. 
i liter = iooo cubic centimeters, cc. 

i kilogram = iooo grams, gm. 
i gram = iooo milligrams, mg. 

American Weights and Measures 

i gallon = 8 pints. 

i pint = 1 6 fluidounces. 

i fluidounce = 8 fluidrachms. 

r fluidrachni = 6o minims, 
i avoirdupois pound = 16 avoirdupois ounces. 
i avoirdupois ounce =437^ grains. 
1 apothecaries' ounce = 8 drachms. 
1 apothecaries' drachm = 60 grains. 

Equivalents 
1 meter = 39.370 inches. 

1 liter = 33.815 U. S. fluidounces. 

1 liter = 35.219 Imp. fluidounces. 

1 cubic centimeter = 16.231 U. S. minims. 
1 kilogram = 32.151 U. S. apoth. ounces. 

1 kilogram = 35.274 avoirdupois ounces. 

1 gram = 15.432 grains. 

1 U. S. fluidounce = 29.57 cubic centimeters. 
1 Imp. fluidounce = 28.39 cubic centimeters. 
- 1 U. S. apoth. ounce = 31.103 grams. 
1 avoirdupois ounce = 28.349 grams. 
1 grain = 64.798 milligrams. 

ijU. S. fluidounce of water weighs 0.95 U. S. apoth. ounce. 
96^U. S. fluidounces of water weigh 100 avoirdupois ounces. 
1 Imperial fluidounce of water weighs 1 avoirdupois ounce. 
1 U. S. gallon = 3.785 liters. 

1 avoird. pound = 453.59 grams. 
1 kilogram = 2.204 avoir, pounds. 

1 pint = 473.179 cubic centimeters. 



224 



APPENDIX 



Table of Approximate Atomic Weights 

(O = 1 6 as basis) 



Name. 



Aluminum. 
Antimony. , 
Arsenic. . . 
Barium . . . 
Bismuth . . 
Boron . . . . 
Bromine . . 
Cadmium . , 
Calcium . . 
Carbon. . . . 
Chlorine . . 
Chromium , 
Cobalt. . . . 
Copper . . . 
Fluorine . . 

Gold 

Hydrogen . 
Iodine . . . . 

Iron 

Lead 

Lithium . . 



1 

Symbol 
Al 


Atomic 
Weight 


27.I 


Sb 


120.4 


As 


75-o 


Ba 


E37-4 


Bi 


208.1 


B 


11. 


Br 


So.o 


Cd 


in. 9 


Ca 


40.1 


C 


12.0 


CI 


35-5 


Cr 


52.1 


Co 


53.9 


Cu 


63.6 


F 


19.0 


Au 


197.2 


H 


1.0 


I 


126.9 ! 


Fe 


56.O 


Pb 


206.9 


Li 


7.0 



Name. 



Magnesium 
Manganese 
Mercury . . 
Molybdenum 
Nickel . . . 
Nitrogen . . 
Oxygen . . 
Phosphorus 
Platinum. . 
Potassium . 
jSelenium . 
Silicon . . . 
Silver . . . 
! Sodium . . 
iStrontium . 
Sulphur . . 
Tellurium . 
Thallium . 
Tin .... 
I Uranium . 
! Zinc .... 



Symbol '£°™* 
Y\ eight 



Kg 

Mn 

Hg 
Mo 
Ni 
N 

O 
P 

Pt 

K 

Se 

Si 

Ag 

Na 

Sr 

S 

Te 

Th 

Sn 

U 

Zn 



24-3 

55-0 

200.0 

96.0 

58.7 
14.0 
16.0 
31.0 

194-9 
39-i 
79.0 
28.4 

107.9 
23.0 
87.6 
32.1 

127.5 
204.1 
119. o 
239.6 
65-4 



APPENDIX 



225 



Volume and Specific Gravity of Water of Different 
Temperatures 

Calculations of Volkmann from results of Kopp, Hagen, 
Matthiessen, Jolly, and Pierre. 



Temperature C. 



9 
10 . 
11 
12 . 

13 
14 . 

15 
16 . 

17 
18 . 

19 
20 . 
21 
22 . 



Specific gravity, | 
or weight of 1 Differences for i°, 
cc. of water in sp. gr. and volume 

vacuo in grams. | 



Volumes of one 

gram of water 

in cc. 



24 ... . 

25 . . . . 



O.99988 
O.99993 
O.99997 
O.99999 
I. OOOOO 
O.99999 
O.99997 
O.99993 
O.99988 
O.99982 
O.99974 
O.99965 

o-99954 
0.99943 
0.99930 
0.99915 
0.99900 
0.99884 
0.99866 
0.99847 
0.99827 
0.99807 
0.99785 
0.99762 
0.99739 
0.99714 
•0.99577 



0.00005 
0.00004 
0.00002 
0.0000 1 
0.0000 1 
0.00002 
0.00004 
0.00005 
0.00006 
0.00008 
0.00009 
o. 0001 1 
0.000 1 1 
0.00013 
0.00015 
0.00015 
0.00016 
0.00018 
0.00019 
0.00020 
0.00021 
0.00022 
0.00023 
0.00023 
0.00025 
0.00027 



1. 00012 
1.00007 
1.00003 
1. 0000 1 

I. OOOOO 

1. 0000 1 
1.00003 
1.00007 
I. OOOI2 
1. 00018 
1.00026 

1.00035 

1.00046 

1.00057 

1.00070 
1.00085 

I.OOIOO 

1.00116 

1. 00134 
I. 00153 

1. 00173 

1. 00194 
1. 00216 
1.00238 
1.00262 
1.00287 

1.00425 



15 



226 APPENDIX 

List of General Reagents and Test Solutions 

Acid, sulphuric (strong). — The commercial acid is sufficient 
for most purposes, where a strong acid is called for. Where 
the pure acid is required it is mentioned in the text. 

Acid, sulphuric (dilute). — Add i part of the above acid to 
4 parts of distilled water, mix thoroughly, and allow to 
stand twenty-four hours. Then siphon, or pour off the clear 
liquid, which is ready for use. The strong acid must be poured 
into the water very slowly, and with constant stirring, to 
avoid a too sudden elevation of temperature. 

Acid, nitric (strong). — The strong commercial acid can be 
employed in most cases where this acid is called for. A pure 
strong acid is also emplo3'ed, occasionally. 

Acid, nitric (dilute). — Where this acid is called for as a rea- 
gent it should be made by mixing i part of the pure strong 
acid with 4 parts of distilled water. It should be free from 
traces of chlorine and sulphates. 

Acid, hydrochloric (strong). — The yellow commercial acid is 
largely used in the laboratory in the preparation of other sub- 
stances. It is seldom pure enough to be employed as a test 
reagent. 

A colorless acid, free from organic matter, iron and traces 
of sulphates, must be used when the pure strong acid is called 
for. 

Acid, hydrochloric (dilute). — This is frequently used in lib- 
erating hydrogen sulphide, carbon dioxide, and hydrogen, 
and for other purposes, and need not be pure. Where the di- 
lute acid is called for as a reagent it must be made by mixing 
1 part of the pure strong acid with 4 parts of distilled water. 

Acid, acetic. — Mix 1 part of the pure "glacial " acid with 
4 parts of water. 

Ammonia water. — The strong solution is seldom used in 
analysis. The solution usually employed is made by mixing 
1 volume of the stronger ammonia water (containing about 
28 per cent, of the gas) with 3 volumes of distilled water. 



APPENDIX 227 

The solution should be free from carbonic acid, as presence 
of this would interfere with several of the tests where it is em- 
ployed. 

Ammonium carbonate. — Dissolve 1 part of the pure pow- 
dered crystals in 5 parts of dilute ammonia water. 

Ammonium chloride. — Dissolve 1 part of the pure salt in 
10 parts of water. 

Ammonium molybdate. — A solution of this salt is chiefly 
used as a test for phosphoric acid, and should be prepared in 
this way : Dissolve 3 grams of the crystals in 20 cc. of water, 
and pour this solution in 20 cc. of strong nitric acid. Warm 
the mixture to about 40 C. (not above), and allow to settle. 

As the reagent does not keep well, it should be made only 
in small quantities. 

Ammonium oxalate. — Dissolve 1 part of the pure crystals 
in 20 parts of water. 

Ammonium sulphide. — Dilute a quantity of strong ammonia 
water with an equal volume of water. Take three-fifths of 
this mixture and saturate it with hydrogen sulphide. Then 
add the remaining two-fifths of the diluted ammonia water 
and mix thoroughly. 

Barium chloride. — Dissolve 1 part of the crystals in 10 
parts of water. 

Calcium hydroxide. — Slake pure white lime and pour over it 
a large excess of water. Allow to settle and throw away the 
clear liquid. Again add pure water, shake thoroughly, allow 
to settle as before, and decant the clear liquid into bottles for 
use. These bottles must be tightly stoppered. The portion 
rejected contains small amounts of impurities, possibly pres- 
ent in the lime. 

Lead acetate. — One part of the pure c^stals to 10 parts of 
w r ater. It may be necessary to add a few drops of acetic acid 
to secure a clear solution. 

Lead acetate, basic. — To make a liter of solution of this re- 
agent weigh out 170 grams of lead acetate and 120 grams of 
yellow lead oxide. 



228 APPENDIX 

Dissolve the lead acetate in 800 cc. of boiling, distilled 
water, in a glass or porcelain vessel. Then add the oxide of 
lead and boil for half an hour, occasionally adding enough 
hot distilled water to make up the loss by evaporation. Re- 
move the heat, allow the liquid to cool, and add enough dis- 
tilled water, previously boiled and cooled, to make the prod- 
uct measure 1000 cc. Finally, filter the liquid in a covered 
funnel. 

Solution of basic lead acetate should be kept in well-stop- 
pered bottles. 

Magnesia mixture. — Dissolve 100 grams of magnesium sul- 
phate and 100 grams of ammonium chloride in 800 cc. of wa- 
ter, and add 100 cc. of strong ammonia water. Allow the 
mixture to stand twenty-four hours, and filter. 

Mercuric chloride. — One part of the pure crystals to 20 parts 
of water. 

Millon's reagent. — Dissolve 1 part of mercury in 2 parts 
of strong nitric acid, by aid of heat finally, and after cooling 
dilute the solution with twice its volume of water. 

Potassium chromate. — Dissolve 1 part of the crystals, free 
from chlorine, in 10 parts of water. 

Potassium dichromate. — Dissolve 1 part of the pure crys- 
tals in 10 parts of water. The dry powdered crystals are 
also used. 

Potassium ferrocyanide. — Dissolve 1 part of the crystals in 
20 parts of water. 

Potassium hydroxide. — Several solutions are employed in an- 
alytical chemistry. For most purposes one containing 10 per 
cent, of the " stick " hydroxide is sufficient. 

Sodium hydroxide. — Dissolve 1 part of the best "stick" 
hydroxide in 10 parts of water, allow to settle, and decant 
the clear solution. 

This solution acts on glass bottles, and soon deposits a 
sediment. Hence, a great deal of it should not be made at 
one time. The glass stoppers of bottles containing sodium 
hydroxide, and many other substances, should be covered 



APPENDIX 229 

with a thin layer of paraffin, which prevents their sticking 
fast. 

Sodium hypochlorite. — The ' ' chlorinated soda ' ' of the U. S. P. 
is to be used here, and the solution may be made in this man- 
ner : Weigh out 75 grams of good commercial ' ' chloride of 
lime " and rub it up with 200 cc. of water to a thin cream. 
Allow this to settle and pour the liquid through a filter. Stir 
the residue with a second 200 cc. of water, pour the whole on 
the filter and wash the insoluble residue with 100 cc. of water, 
allowing this to mix with the 400 cc. Now dissolve 150 
grams of sodium carbonate crystals in 300 cc. of hot water 
and pour this into the other solution. Warm the mixed so- 
lution and stir it well. Pour the mixture on a filter and when 
the liquid has run through pour on water enough to bring 
the filtrate up to 1000 cc. The solution so obtained should 
be kept in the dark. 

Special Reagents 

Solutions for Sugar Tests 
Fehling solution. — This has been referred to at length in 
Chapter III, and its preparation will now be given. In pres- 
ence of alkali, copper solutions are reduced by dextrose ap- 
proximately according to this proportion : 

5(CuS0 4 . 5 HO):C 6 H i2 6 . 
1248.00 : 180 

from which it follows that 34.66 mg. of the crystallized sul- 
phate in solution oxidizes 5 mg. of dextrose in solution. 

The Fehling solution proper consists of a mixture of copper 
sulphate, an alkali, and a tartrate. Investigation has shown 
that alkali sodium hydroxide is preferable and that Ro- 
chelle salt is the best tartrate for the purpose. It has also 
been found that the best results are obtained if the copper and 
tartrate are mixed just before needed for use. Therefore, pre- 
pare solutions separately as follows : 

1. Dissolve 69.32 grams of pure, recrystallized copper sul- 
phate in distilled water to make 1 liter of solution. Much of 



230 APPENDIX 

the copper sulphate sold by druggists contains ferrous sul- 
phate and is not suitable for the purpose. 

2. Dissolve 100 grams of sodium hydroxide in sticks in 
500 cc. of water, heat to boiling and add gradually 350 grams 
of pure recrystallized Rochelle salt. Stir until all is dissolved. 
Allow the solution to stand twenty-four hours in a covered 
vessel, then filter through asbestos into a liter flask and add 
water enough to make the solution 1 liter. The sodium hy- 
droxide for this purpose should be the grade designated "pre- 
cipitated by alcohol ' ' and the Rochelle salt should be prac- 
tically pure. 

3. By mixing equal volumes of these two solutions the 
Fehling liquid is prepared, containing 34.66 mg. of the cop- 
per sulphate in each cubic centimeter. This mixture is made 
when required for use. 

The Loewe solution. — According to Loewe, copper solutions 
prepared with glycerol are more stable than are those with a 
tartrate, while the oxidizing power of the copper hydroxide 
is practically the same. For quantitative tests Loewe pre- 
pares a solution by mixing 

Crystallized copper sulphate . . . 34.66 gm. 

Pure glycerol . . . . 26.00 gm. 

Sodium hydroxide solution, L34sp.gr. . 70.00 cc. 

with a small amount of water and heating to dissolve, after 
which the solution is diluted to 1 liter. 

When used as a qualitative test this solution is diluted by 
adding glycerol. In place of the crystallized copper sulphate 
Loewe recommends to weigh out the corresponding amount 
of precipitated, washed, and dried copper hydroxide, which 
can be kept indefinitely in perfectly stable form. Haines' 
solution is a modification of the Loew r e solution containing a 
relatively large amount of glycerol and alkali, and is em- 
ployed in qualitative testing. 

Pavy's solution. — The use of the Pavy liquid as a sugar test 
was explained in Chapter III. It is prepared as follows : 

Dissolve 34.66 grams of crystallized copper sulphate, 170 



APPENDIX 231 

grams of Rochellesalt, and 170 grams of good stick potassium 
hydroxide in distilled water to make 1 liter. Mix 120 cc. of 
this solution with 400 cc. of strong ammonia water, sp. gr. 
0.88, and dilute with distilled water to 1 liter. The oxidiz- 
ing power of this solution is assumed to be just one- tenth of 
that of the Fehling solution, which would follow if the reac- 
tion takes place in the proportion 6(CuSO .5H 2 0):C 6 H io 6 . 

Loewe-Pavy solution, as recommended by Dr. Purdy. 

This is made by dissolving 

Crystallized copper sulphate . . .4.75 gm. 
Potassium hydroxide . . . . 23.50 gm. 

Ammonia water, sp. gr. 0.90. . . 350.00 cc. 

Glycerol ...... 38.00 cc. 

in enough water to make 1000 cc. 

Dissolve the copper sulphate and glycerol in 200 cc. of the 
water. In another 200 cc. dissolve the alkali. Mix the two 
solutions, cool, add the ammonia and make up to 1000 cc. 
Pure chemicals must be employed. 

According to Dr. Purdy 35 cc. of this solution oxidize 20 
mg. of dextrose. But the oxidizing power of the solution 
depends on the amount of ammonia and fixed alkali present 
and on the concentration of the sugar solution. 

In the Pavy and Purdy liquids the oxidizing power is 
apparently assumed to be independent of the strength of the 
sugar solution added. Pavy and Purdy assume the factor 
8.316 CuS0 4 .5H 2 to iC 6 H i2 6 . But Peska has shown that 
the factor varies over 2 per cent with solutions ranging from 
0.1 to 1 percent in strength. 

As in practical work it is desirable to employ a solution, 
1 cc. of which oxidizes some simple unit amount of sugar, 
the author has been using lately one with a value of 1 cc. for 
each milligram of sugar oxidized in 0.2 per cent, solutions. 
It is made with the following amounts per liter : 

Copper sulphate, cryst - - - 8.166 gm. 

Sodium hydroxide (100 per cent.) - 15.000 gm. 

Glycerol ------ 25.000 cc. 

Ammonia water, 0.9 sp. gr., - - 350.000 cc. 
Water to make - 1,000.000 cc. 



232 APPENDIX 

Of this solution use 50 cc. and dilute with water to 100 ec. 
To prevent too rapid an escape of ammonia and avoid reoxi- 
dation to some extent, add to the mixture, while warming, 
enough pure white solid paraffin to make a layer 3 or 4 milli- 
meters in thickness when melted. The burette tip for 
discharging the sugar solution or urine is made long enough 
to pass down the neck of the flask and below this paraffin. 
By boiling gently and adding the weak saccharine liquid 
slowly, very close and constant results may be obtained. At 
the end of the titration the paraffin is solidified 03- inclining 
the flask and immersing it in cold water, or by flowing cold 
water over it. The reduced liquid is then poured out and the 
cake of paraffin is thoroughly washed for the next test. A 
flask so prepared may be used for a hundred titrations. The 
solid paraffin is much preferable to the oil recommended by 
Allen and Peska. To prevent bumping and facilitate easy 
and uniform boiling, it is well to add a few very small frag- 
ments of pumice-stone. A solution made as above is not too 
strong in copper for accurate work, but the volume of 
ammonia necessary to hold a much larger amount of the 
reduced oxide in solution would render the process very 
inconvenient. 

Schmiedeberg's solution. — Dissolve 34.66 grams of pure crys- 
tallized copper sulphate in 200 cc. of water and 16 grams of 
mannitol in 100 cc. of water. Mix the two solutions and add 
480 cc. of sodium hydroxide solution, having a specific gravity 
of 1. 145. Dilute to 1 liter. This solution is assumed to have 
the oxidizing power of the Fehling solution. 

Knapp's solution. — This is made by dissolving 10 grams of 
dry mercuric cyanide in 600 cc. of distilled water. To this 
solution is added 100 cc. of a solution of sodium hydroxide, 
having a specific gravit}- of 1.145, an d the mixture is diluted 
to 1 liter. 10 cc. of this solution may be taken as sufficient 
to oxidize 25 mg. of dextrose in dilute solution. In strong 
solution the oxidizing power is less. The test should be made 
with a dilution as indicated in the text. 



APPENDIX 233 

Sachsse's solution. — Dissolve 18 grams of pure mercuric 
iodide and 25 grams of potassium iodide in water. Add a 
solution of 80 grams of good potassium hydroxide and dilute 
to 1 liter. It has been shown that 50 cc. of Sachsse's solu- 
tion is reduced by 168 mg. of dextrose, in a solution of about 
1 per cent, strength. 

Fron's reagent. — Dissolve 7 grams of potassium iodide in 
20 cc. of water ; heat and add 1.5 grams of bismuth subnitrate 
and 1 cc. of strong pure hydrochloric acid. 

Indicators 

The indicators most commonly employed in the titration 
of acids and alkalies are aqueous or alcoholic solutions of 
litmus, cochineal, phenol-phthalein, methyl orange, and roso- 
lic acid. In addition to these, certain others are emplo3 r ed for 
special purposes, and among them there may be mentioned, 
congo red, benzopurpurin, methyl violet, and the tropceolins. A 
few explanations will be given on the preparation of the first. 

Litmus. — Crude litmus comes in commerce in the form of 
small blue cubes. These are powdered and extracted by hot 
water. The aqueous solution is concentrated and acidified 
with acetic acid, after which it is evaporated to a paste. Treat 
this with an excess of 85 per cent, alcohol which dissolves 
foreign matters but leaves the true color. Throw the mix- 
ture on a filter and wash the residue with strong alcohol. 
Then dissolve the precipitated color on the filter by means of 
hot water and keep this aqueous solution for use in a bottle 
loosely stoppered, as access of air is necessary for its preser- 
vation. 

A neutral litmus solution is used for certain purposes and 
may be prepared by dividing the aqueous solution just de- 
scribed into two portions, one of which is rendered faintly 
acid by nitric acid, while the other is made alkaline by potas- 
sium hydroxide, added in drops of very dilute solution. On 
mixing these two liquids the product will be found practically 
neutral. 



234 APPENDIX 

Cochineal. — A solution is made by extracting i part of 
the crushed cochineal with 10 parts of weak alcohol. This 
indicator is valuable in titrating in presence of carbonic acid 
or ammonia. 

Phenol-phthalein. — Dissolve i part of the pure commer- 
cial product in 200 parts of 50 per cent, alcohol. This indi- 
cator cannot be well used with ammonia or in presence of 
free carbonic acid. 

Methyl orange. — Dissolve 1 part of the color in 1000 parts 
of distilled water. Although this solution is very weak a 
single drop is sufficient for an ordinary titration ; with more 
the change of color is less characteristic or sharp. The indi- 
cator is valuable in the titration of carbonates or ammoniacal 
liquids. Carbonic acid does not act on it. 

Rosolic acid. — Dissolve 1 part in 500 parts of 50 per cent, 
alcohol. This is a sensitive indicator for the mineral acids. 

Some Volumetric Solutions 

Standard sulphuric acid. — This is required for the determina- 
tion of ammonia and for the determination of alkalinity of 
urine as given in the text. The preparation of the ' ' normal ' ' 
acid, 49.05 grams of H 2 S0 4 to the liter, will be given first. As 
the strongest obtainable commercial acid always contains 
some water, it is not practicable to weigh out 49.05 grams 
and dilute it to one liter. The solution may be made accurately 
by a less direct method, as follows : Measure out 30 cc. of 
pure strong acid and pour it into 900 cc. of distilled water, 
with stirring; when the mixture cools dilute it to one liter, 
which gives a solution a little too strong, and find the actual 
strength in this manner. Weigh out 2.65 grams of pure, per- 
fectly dry sodium carbonate and dissolve it in a little water, 
in a beaker. Add two drops of a w r eak solution of methyl 
orange as indicator and then from a burette run in some of 
the sulphuric acid, very slowly, and with constant agitation 
of the beaker liquid, until the color changes to a faint pink. 



APPENDIX 235 

At this stage just enough acid has been added to neutralize 
the carbonate according to the equation : 

H SO, + Na o CO, == Na SO, + HO + CO 

24' 23 24' 2' 2 

98.1 -f 106 = 142. 1 + 18+44 
The 2.65 grams of pure carbonate taken will neutralize 2.4525 
grams of pure acid and therefore this weight of acid must be 
present in the volume added from the burette. It is also the 
weight of acid which should be present in 50 cc. of the nor- 
mal solution. If the experiment shows that we have used 
48 cc. of the acid in the test with the carbonate it remains to 
simply dilute with pure water, to bring each 48 cc. up to 50, 
If we have left 900 cc. we may dilute according to this pro- 
portion : 

48 : 50 :: 900 : x, x — 937.5 

Therefore, we add 37.5 cc. of water to the 900 cc. to secure 
the right volume. 

From this normal solution a tenth-normal, N/10, may be 
made by diluting 50 cc. to 500 cc, and any desired solution, 
below the normal, may be made in a corresponding manner. 

Standard sodium hydroxide solution. — A fifth normal, N/5, or 
tenth normal, N/10, alkali solution is employed in measuring 
the acidity of the urine and in titrating the excess of acid 
used in the ammonia determination. The preparation of a 
fifth-normal solution will be given here. This contains 8 
grams of actual NaOH to the liter and must be made indi- 
rectly as the commercial hydroxide is never of definite 
strength. A solution of slightly greater than the desired 
strength is first made and tested, after which water is added 
in proper amount to bring to the standard dilution. The 
value of the alkali solution may be found by comparison with 
the standard sulphuric acid, as described above. 

Weigh out 10 grams of good stick sodium hydroxide, dis- 
solve in water, and dilute to one liter. Measure into a beaker, 
accurately, 50 cc. of tenth-normal sulphuric acid and add a 
few drops of weak phenol-phthalein solution as indicator. 
Heat to boiling, and from a burette run into this, gradually, 



236 APPENDIX 

the alkali solution and continue the addition until a perma- 
nent faint red or pink color persists, showing the neutraliza- 
tion of the standard acid by the alkali. If the alkali were of 
exactly fifth-normal, N/5, strength 25 cc. would be required 
for this, but as the solution was made a little strong less will 
be needed. If 23 cc. is sufficient to neutralize 50 cc. of the 
N/10 acid, each volume of the 23 cc. must be diluted to 25 cc. 
with water to produce the required solution. 900 cc. of the 
alkali should be diluted by this proportion : 

25 : 25 :: 900 : x. x = 978.3 
Instead of comparing the alkali solution with the standard 
sulphuric acid a very convenient and accurate method is to 
compare with potassium bitartrate, l ' cream of tartar, ' ' taken 
as a standard. This substance ma}' be obtained in a condi- 
tion of great purity, suitable for weighing out directly. It 
combines with the alkali according to the equation : 
KHC HO,- NaOH = KXaC HO.-HO 

446 4 4 6 ' 2 

188. 1 40 = 210. 1 18 

Weigh out 0.9405 gram of the bitartrate, dissolve in 50 to 100 
cc. of water, add a little phenol-phthalein, heat to boiling, and 
run in the alkali solution jmtil the pink color appears. The 
dilution is made as before. From the equation it appears 
that the amount of bitartrate taken corresponds to 25 cc. of 
N/5 solution of alkali. 

By diluting 500 cc. of the N/5 alkali solution to 1000 cc. 
with distilled water a solution of N/10 strength is obtained. 

Standard permanganate solution. — In the titration of uric 
acid a solution of potassium permanganate of N/20 strength 
is employed. As this substance ma}- be obtained in pure con- 
dition the standard solution may be made by weighing out, 
accurately, 1.581 grams, dissolving in distilled water, and di- 
luting to 1 liter. It is customary to control the strength 
of permanganate solutions by titration with ferrous solutions, 
but for the purpose of this book that is not necessary. 

Standard silver nitrate solution. — This is used in the titra- 
tion of chlorides in urine and is generally of N/10 strength. 



APPENDIX 237 

Dissolve 16.99 grams of the pure dry crystallized nitrate in 
distilled water and dilute to 1 liter with distilled water. 

This is used in neutral solution with a ehromate as indi- 
cator. When the Volhard method is employed an- acid solu- 
tion of the nitrate may be very conveniently made by dissolv- 
ing 10.79 grams of pure silver foil in nitric acid in a flask. 
The excess of acid is removed by evaporation, a current of 
air is blown through to drive out nitrous fumes, and then dis- 
tilled water is added to bring the volume up to 1 liter. 

Standard thiocyanate solution. — This is employed in N/10 
strength in the titration of chlorides by the Volhard method, 
and in N/50 strength in the titration of uric acid by the Hay- 
craft method. The N/10 solution contains 7.61 grams of the 
salt in a liter and is made by weighing out about 8 grams of 
the crystalline commercial product, dissolving to make a liter 
of solution and then titrating against the standard N/10 sil- 
ver solution as described in the text. The N/50 solution is 
made from this by dilution. 

Ferric alum, indicator. — This is used with the above solu- 
tions and is simply a strong solution of ammonium ferric sul- 
phate in water. The salt must be perfectly free from chlorine. 

Standard mercuric nitrate solution. — This is employed in the 
titration of urea by the method of Liebig, and is prepared by 
the process as described in the text. 

Ehrlich's reagent. — This is applied in the so-called diazo re- 
action and is made in this manner : Dissolve 1 gram of sul- 
phanilic acid in 200 cc. of water with the addition of 10 cc. of 
pure strong hydrochloric acid. Make a second solution by 
dissolving 1 gram of sodium nitrite in 200 cc. of water. The 
reagent for actual use is prepared by mixing 50 cc. of the first 
solution with 5 cc. of the second. The mixture is added to 50 
cc. of the urine to be tested, and then ammonia enough to 
give a strong alkaline reaction. The sulphanilic acid and 
nitrite react on each other in this way : 
C 6 H .HS(X .NH + NaN0 2 + HC1 = 

3 C 6 H 4 .N .S0 3 + NaCl + 2HO. 

The active agent is the diazobenzenesulphonic acid, 



2 3 8 



APPENDIX 



C 6 H 4 .N 2 .S0 3 , which is sometimes employed directly. But, 
as it is not very stable, it is best prepared in solution by the 
reaction given. 

Phosphotungstic acid. — Dissolve ioo grams of crystallized 
sodium tungstate and 25 grams of glacial phosphoric acid in 
500 cc. of water by aid of heat. Add 50 cc. of pure diluted 
hydrochloric acid. 



Tables Illustrating the Characteristics of Normal and 
Pathological Urine 



Quantity 

Color, 

Reaction, 

Specific gravity, 

Total solids, . 

Urea, 

Uric acid, 

Sugar, 

Albumin, 

Diazo reaction, 

Indican, 

Chlorides, NaCl 

Phosphates, . 

Sulphates, . 

Deposit, 



B 



A. Normal Urine 

1000 to 2000 cc. 

Straw-yellow. 

Acid. 

1.005 to 1.030. 

16 to 80 grams daily. 

15 to 70 grams daily. 

0.2 to 1 gram daily. 

None. 

None. 

Absent or very weak. 

Present normally in traces. 

5 to 25 grams daily. 

2 to 5 grams of P 2 O s . 

About 2 grams daily. 

Normal^ slight in fresh urine. 

Clinical Significance 



Quantity — Increased largely in diabetes mellitus and dia- 
betes insipidus. Increased also in chronic in- 
terstitial nephritis and in amyloid kidney. 
Decreased in passive hyperemia, acute neph- 
ritis, and generally in chronic parenchym- 
atous nephritis. 



APPENDIX '239 

Color. — Light in diabetes mellitus and diabetes insipidus, 
also in chronic interstitial nephritis, chronic 
diffuse nephritis and amj'loid kidney. Usually 
darker in chronic parenchymatous nephritis, 
in passive hyperemia, and acute nephritis. 
Usually darker in high fever; in jaundice a 
peculiar greenish j-ellow. Bloody urine is 
reddish brown. Many drugs impart color to 
be recognized only by special tests. 

Reaction. — Acid normally and in fevers and diabetes. Nor- 
mal urine becomes alkaline on standing from 
fermentation. It is usually alkaline in chlo- 
rosis and pernicious anemia. Urine may show 
an ammoniacal reaction from introduction of 
the ferment into the bladder by a dirt}* cathe- 
ter. 

Specific gravity. — Low in diabetes insipidus and in chronic 
interstitial nephritis. It is especially high in 
diabetes mellitus, and high in passive hypere- 
mia and acute nephritis. Large variations 
naturally follow greatly increased or dimin- 
ished consumption of liquid. 

Total solids. — Increased in gross amount in diabetes melli- 
tus and insipidus, but generally diminished 
in nephritis, chronic and acute, although with 
scanty discharge the specific gravity may be 
high. 

Urea. — In total amount increased in diabetes mellitus and 
insipidus, but generally diminished in differ- 
ent kinds of nephritis. In acute nephritis and 
passive hyperemia, with diminished quantity 
of urine, the percentage amount of urea may 
be increased. 

Uric acid. — Generally increased in fevers. Bears normally 
a fairly definite relation to amount of urea, 
but clinical significance is often obscure. 



240 APPENDIX 

Sugar. — Diabetes mellitus. A small trace of sugar appears 
to be normally present in urine, but this is 
not recognizable with the ordinary test. Tem- 
porarily, however, sugar ma}- be present with- 
out diabetes ; it is the continued presence of 
the substance which is here characteristic and 
important. 

Albumin. — Normally absent. Its presence is usually sug- 
gestive of disease of kidney. The continued 
appearance of large amounts of albumin is 
always pathological. But temporarily, even 
more than traces ma}' appear without connec- 
tion with disorders of the kidney, as in the 
albuminuria of pregnancy, and in albuminuria 
in which the albumin is derived from parts of 
the tract below the kidney. 

Globulin. — Significance in general the same as that of al- 
bumin. 

Peptone. — Generally pathological and suggestive of sup- 
purative changes somewhere. 

lndican. — Normally present in small amount, but may be 
greatly increased in diabetes mellitus or in 
diseases accompanied by marked putrefactive 
changes in the intestines. 

Diazo reaction. — Very weak in normal urine but usually 
sharp in fever urine. 

Chlorides. — Normally always present, but great variations 
depend on character of food. May be greatly 
decreased in pleurisy and acute pneumonia. 

Sulphates. — Always present in traces. An increase in the 
ethereal sulphates is pathological, and accom- 
panies intestinal diseases as a result of putre- 
factive changes. 

Phosphates. — Normally always present, and variable with 
diet. Increased in osteomalacia and similar 
disorders. 



APPENDIX 241 

Sediment. — Normally is slight in fresh urine, but usually 
becomes abundant, if the urine be allowed to 
stand, from putrefactive changes. In the fol- 
lowing tables the characteristics of certain sed- 
iments are given. 

C. Diabetes Meelitus 

Quantity, Very much increased, may amount 

to 15 liters, in 24 hours. 

Color, Pale yellow. 

Reaction, Acid. 

Specific gravity, . . . High usually, may reach 1.060. 
Total solids, Increased in total amount, but 

lower in percentage. 
Urea, Increased in total, but percentage 

low. 
Sugar, Always present, and from 100 to 

900 grams, in 24 hours. 

Albumin, Usually absent. 

Acetone and ace- ) 

. , > . . . Usually present. 
to-acetic acid, ) J r 

Indican, Usually increased. 

Phosphates, Increased. 

Sulphates, Increased. 

Deposit, Little. 

Microscopic examination, Negative. 

D. Diabetes Insipidus 

Quantity, Greatly increased. 

Color, Very light, clear. 

Reaction, Neutral or feebly acid. 

Specific gravity, . Very low. 

Total solids, Increased in total amount, but 

low in per cent. 

Urea, Increased in total amount, per- 
centage low. 

Sugar, Absent. 

Albumin, Absent. 



242 APPENDIX 

Deposit, None. 

Microscopic examination, Negative. 

E. Chronic Parenchymatous Nephritis 
(Chronic Bright 's Disease) 

Quantity, Usually much below normal. 

1000 cc. or less. 

Color, Dark, sometimes described as 

smoky, turbid. 

Reaction, Acid. 

Specific gravity, Normal or higher. 

Urea, Below normal. 

Albumin, Abundant. 

Sediment, Heavy. 

Microscopic examination. Hyaline casts, granular casts, 

epithelial casts, fatty casts, red 
blood corpuscles, leucocytes, so- 
dium and ammonium urates. 

F. Acute Nephritis 
(Acute Bright \s Disease) 

Quantity, Scant}', often below 500 cc. 

Color, Dark, smoky, turbid, appearance 

largely due to blood. 

Reaction, Usually acid. 

Specific gravity, .... High. 

Total solids, Lessened. 

Urea, Total quantity much diminished 

but per cent. high. 

Albumin, Much present. 

Sediment, Much. 

Microscopic examination, . Blood corpuscles, leucocytes, 

blood corpuscle casts, epithelial 
casts, granular casts, hyaline 
casts, uric acid, urates, and" renal 
epithelium. 



APPENDIX 243 

G. Chronic Interstitial Nephritis 
(Primary Contracted Kidney) 

Quantity, Usually excessive, 2000 to 4000 

cc. frequently. 

Color, .... . . Light. 

Reaction, Acid. 

Specific gravity, . . . Low, often 1.005. 

Total Solids, Diminished. 

Urea, Much diminished in total quan- 
tity and per cent. 

Albumin, Little or none at all, but under 

temporary renal congestion it 
may be considerable. 

Sediment, Little. 

Microscopic examination, . Hyaline casts, granular casts, but 

not always present, blood cor- 
puscles absent. 

H. Chronic Diffuse Nephritis 

(Secondary Contracted Kidney) 

Quantity, Normal or increased. 

Color, Light and usually clear. 

Reaction, Acid. 

Specific gravity, . . . Below normal. 

Total solids, Diminished. 

Urea, Low. 

Albumin, Present, and often in large 

amount. 

Sediment, Considerable. 

Microscopic examination, . Casts, numerous and of all kinds, 

acid urates. 

I. Amyloid Kidney 

(Not Associated with Nephritis) 

Quantity, Large. 

Color, Pale yellow, clear. 



244 APPENDIX 

Reaction, . . . . . . Acid. 

Specific gravity, .... Normal or diminished. 

Total solids, Diminished. 

Urea, Diminished. 

Albumin, Usually abundant, accompanied 

by globulin. 

Deposit, Absent or small. 

Microscopic examination, Often negative. A few waxy or 

hyaline casts may be present. 

J. Passive Hyperemia 

Quantity, Scanty. 

Color, Dark, sometimes bloody in ap- 
pearance. 

Reaction, Acid. 

Specific gravity, High. 

Total solids, Usually diminished in total 

amount, but in percentage in- 
creased. 

Urea, Total decreased, but percentage 

may be increased. 

Albumin, Present, but variable in amount. 

Deposit, Generally considerable. 

Microscopic examination, . Hyaline casts, red blood corpus- 
cles, uric acid, and urates. 



INDEX 



Abnormal colors 92 

Acetoacetic acid 48 and 84 

Acetone 48 and 81 

Adenin 171 

Accidental albuminuria 17 

Acid albumin 19 

fermentation 201 

hippuric 117 

phosphate 145 

Acids from bile 102 

reagents 226 

Acidity, determination 12 

Acute nephritis 242 

parenchymatous nephritis . 17 

yellow atrophy 9 

Albumin 24.0 

Albumins 16 

Albuminometer 32 

Albumose 37 

Aldehydes 84 

Alkali albumin 19 

Alkalinity, determination 12 

Alkaline carbonates 10 

Alkali phosphates 148 

Almen's test 99 

Ammonia 167 

copper tests 72 

determination of . . . .168 

Ammonium carbonate . . .11 and 167 

and urea . . 121 

chloride method . . .114 

sulphate 39 

thiocyanate 111 

urate 203 

Amorphous phosphates 213 

Amount of chlorides 154 

phosphates 148 

sugar 67 

sulphates 163 

urea 120 

uric acid 105 

xanthin 172 

Amphoteric reaction 10 

Amyloid kidney 243 



Analyses of urine 3 and 5 

Analysis of calculi 216 

a-Naphthol test 63 

Appendix 223 

Atomic weights 224 

Bacilli in urine 198 

Bacteria in urine 196 

Baryta solution 126 

Benzoyl amidoacetic acid 117 

Bile acids 87 and 102 

Biliary calculi ^08 

colors 92 

Bismuth test 58 

Biuret test 39 and 42 

Blood casts. 189 

colors 90 

corpuscles 180 

tests 98 

Boettger's test 58 

jS-oxybutyric acid 86 

Bread diet 5 

phosphates in 146 

Bright's disease 17 and 242 

Bunge's tables of urine composi- 
tion 5 

Calcium carbonate 209 

oxalate 209 

phosphates . . . .145 and 212 

sulphate 209 

Calculi 201 

analysis of 216 

Cancer tissue 195 

Carbohydrates. . . . ' 81 

Carnin 171 

Carrot colors 102 

Casts 18 and 188 

Centrifugal machines 178 

precipitation 33 

Chautard's test 84 

Chlorides 145 

and urea 129 

in diabetes insipidus . . 154 
Chloroform and sediments .... 176 
Cholesterin 207 



246 



INDEX 



Chromate indicator 157 

Chronic parenchymatous nephritis242 

Chrysophanic acid 100 

Clearing urine 22 

Coagulation tests 19 

Cochineal 234 

Coefficient of Haeser 7 

Color of urine 13 and 239 

and concentration 14 

tests for sugar 75 

Coloring-matters 87 

Columnar cells 1S5 

Conical epithelium 185 

Contracted kidney 243 

Copper sulphate 51 

Correction for chlorides 129 

Creatinin 83, 167 and 173 

Crystallin 35 

Crystallization of hippuric acid . . nx 
Crystals of calcium oxalate .... 210 

hippuric acid 208 

leucin 205 

phosphates 211 

tyrosin 205 

uric acid 202 

Cjdindroids 190 

Cystin 206 

calculi 217 

plates 205 

Daily excretion 7 

Detection of acetoacetic acid ... 84 

acetone 81 

albumin 17 

bile 88 

bile acids 102 

blood 98 

casts 188 

creatinin 173 

indican 90 

sugar 50 

sulphates 165 

Determination of acidity 12 

albumin 30 

alkalinity ... 12 
ammonia .... 168 
chlorides .... 154 
phosphates . . . 145 

sugar 68 

sulphates .... 166 



Determination of urea 123 

uric acid .... 105 
Determinations by polarimetry . . 76 

Dextrose tests 50 

Diabetes 84 

insipidus 241 

mellitus . . . . 8, 48 and 241 

Diacetic acid 84 

Diazobenzene sulphonic acid ... 95 

Diazo reaction 95, 237 and 240 

Diffuse nephritis 243 

Dilution test 36 

Dimethyl xanthin 171 

Doremus' apparatus 143 

Double iodide test 25 

Earthy phosphates 148 

Egg albumin 16 

Ehrlich's reagent 237 

test 95 

Epithelium casts 189 

cells 184 

from different sources 185 

Erythro dextrin 81 

Esbach method 32 

Ethereal sulphates 163 

Excretion, daily 7 

of nitrogen 119 

False casts 189 

Fat globules 208 

Fatty casts 190 

Fehling solution 69 and 229 

test 53 

Flat cells 185 

Fermentation test 64 

Ferric chloride test 85 

sulphate, indicator 160 

Ferrocyanide, indicator 151 

test 27 

Fokker-Hopkins method 114 

Foreign matters in urine 214 

Fron's reagent 59 and 233 

Fruit sugar 79 

Fungi in urine 196 

Gas methods for urea 133 

reduction, urea method . . . .136 

General reagents 226 

Globulin 35 and 240 

Glucose 48 

Glycocoll 119 



INDEX 



247 



Glycosuria 48 

Gmelin's test 93 

Gout 105 

Granular casts 190 

urates 203 

Gravel 202 

Gravimetric albumin test 30 

Guaiacum test for blood 99 

Guanin 171 

Haeser's coefficient 7 

Haines' solution 230 

Hairs and fibers in urine 214 

Haycraft method in 

Heller's tests 94 and 98 

Hematin tannate 98 

Hematuria 97 

Hemialbumose 37 

Hemoglobinuria 97 

Heteroxanthin 171 

Hippuric acid 117 and 209 

Hoffmeister's tests 43 

Hopkins' method 114 

Huefner apparatus 135 

Human spermatozoa 195 

H3^aline casts 190 and 192 

Hypobromite method 133 

Hypochlorite method 133 

Hypoxanthin 171 

Illumination, microscopic 194 

Indican 90, 164 and 240 

Indicators 233 

Indigotin 90 

Indol 164 

Indoxyl 90 and 164 

Inosite 81 

Interstitial nephritis 243 

Intestinal putrefaction and sul- 
phates 164 

Kjeldahl process 144 

Knapp's solution 74 and 232 

L,actose 80 

I,ardacein 18 

L,egal's test 82 

I,eucin 204 

leucocytes 182 

I,evulose 79 

Iyieben's test 83 

Iyiebig's method 123 

fallacies in . . . . 132 



litmus 233 

I,oewe test 57 

I,oewe-Pavy solution 231 

L,oewe solution 230 

I y ugol's solution 83 

Magnesia mixture 108 and 228 

Magnesium phosphate . . 145 and 212 

Meat diet 5 

Melanin 96 

Mercuric iodide test 25 

nitrate 123 

oxide 123 

Mercury tests for sugar 74 

Methemoglobin 97 

Method for sediments 177 

Metludene blue test 66 

Methyl orange 126 and 234 

Methylxanthin 171 

Micrococci in urine 197 

Micrococcus urese 13 

Microscopic examinations 177 

Milk sugar So 

Molds in urine 196 

Molisch's test 63 

Moore's test 50 

Mucin 27, 29 and 46 

bands 187 

Mucus corpuscles 182 

Murexid test 106 

Muscle sugar 81 

Myosin 35 

Nature of sugars 50 

Nephritis 243 

Neutral phosphates 212 

Nitric acid coagulation 20 

Nitrogen excretion 119 

method for urea 133 

Nitroprusside reaction S3 

Normal colors 87 

reaction 9 

urine 3 

Odor of urine 13 

Organic acids 9 

sulphates 166 

Organized sediments 179 

Osteomalacia, phosphates in . . . 147 

Oxidation of uric acid 115 

Oxybutyric acid 48 

Parasites in urine 200 



248 



INDEX 



Paraxanthin 171 

Parkes' table 4 

Passive hyperemia 244 

Pavy's solution 72 and 230 

Peptones 40 and 240 

Permanganate, standard 236 

Pettenkofer test 103 

Pflueger's correction 130 

factor 124 

Phenols 101 

Phenol-phthalein 234 

Phenol poisoning and sulphates . 164 

test 29 

Phenylglucosazone 62 

Pheuylhydrazine test 61 

Phosphate crystals 211 

Phosphates 145 and 240 

determination of . . . . 149 

in foods 146 

Phosphatic beverages 147 

calculi 218 

diabetes 147 

Phosphomolybdic acid 42 

Phosphorus poisoning . . .40 and 204 

Phosphotungstic acid 43 

Physiological acetonuria 82 

albuminuria .... 16 

glycosuria 48 

Picric acid test 28 and 65 

Polarimetry 76 

Potassium permanganate . 115 and 236 

Precipitation test 107 

Preliminary tests 1 

Preservatives of sediments. 1 76 and 193 

Primary calculi 215 

Purdy solution 231 

Pus corpuscles 182 

Putrefactive changes, sulphates in 163 

odor 13 

Pyogenic peptonuria 41 

Qualitative tests 2 

Quantitative tests 3 

Reaction 9 and 239 

Reagents 226 

Recognition of blood 181 

urea 132 

Reduction tests 68 

Renal albuminuria 17 

Rhubarb colors 100 



Rickets, phosphates in 147 

Rosolic acid 234 

Sachsse's solution 74 and 233 

Safranine test 66 

Salicylic acid 11 and 100 

Salkowski-I^udwig method .... 108 

Santonin 100 

Sarkin 171 

Scaly epithelium 184 

Schmiedeberg's solution 232 

test 57 

Secondary calculi 215 

Sediments from urine 175 

Separation of xanthin bodies . . .172 

Serum albumin 17 

vSerum globulin 35 

Silver nitrate solution in 

standard j 236 

solution, volumetric .... 156 

urate , .... in 

Skatol 164 

Sodium bicarbonate in urea test . . 130 
carbonate solution . . . .126 
hydroxide, standard . . . 235 
hypochlorite . . . 134 and 229 

urate 204 

Solids in urine 8 

Solutions for sugar tests 229 

Specific gravity 5 and 239 

rotation 78 

Spectroscopic tests for blood ... 97 

Spermatozoa 194 

Spherical epithelium 184 

Squibb's apparatus 141 

Standard chloride solution .... 157 
mercuric nitrate . . . .125 

silver solution 156 

thiocyanate solution . . 160 

urea solution 126 

Struve's test 98 

Subnitrate test 58 

Sugar 4 8 and 2 4° 

test by polarimetry 76 

Sulphate test 36 

Sulphates 145, 163 and 240 

from albumin 163 

Sulphur in foods 163 

Sulphuric acid, standard 234 

Table of tests 2 



INDKX 



249 



Tables, atomic weights 224 

clinical significance . . . 238 

normal urine 238 

specific gravity of water . 225 
tension of aqueous vapor . 136 
weights and measures . . 223 

Tanret's test 25 

Temporary glycosuria 49 

Tests for acetoacetic acid 84 

acetone 81 

albumins 16 

albumose 3 8 

bile colors 93 

coloring-matters .... 88 
ethereal sulphates ... 165 

globulin 36 

indican 90 

leuc'n and tyrosin .... 206 

mucin 46 

outline of 1 

oxybutyric acid 86 

peptones 42 

preliminary 1 

pus 183 

sugars 50 

uroerythrin 88 

urobilin 88 

urohematin 89 

urophain 89 

Thiocyanate standard 237 

Thudichum's tables 3 and 4 

Titration of uric acid 115 

Total nitrogen 143 

solids 239 

Tribromphenol 102 

Trichloracetic acid test 29 

Triple phosphate 211 

Trisodium phosphate 10 

Trommer's test 51 

Trousseau's test 94 

True albuminuria 17 

Tyrosin 204 

Unorganized sediments . . 180 and 201 
Uranium method for phosphates . 150 
Urate calculi 217 



Urate casts [89 

Urates 21 and 105 

Urea 119 and 239 

apparatus . . 135, 137, 141 and 143 

decomposition 10 

fermentation 11 

in fevers 121 

solution 126 

Uric acid 105, 171, 201 and 239 

precipitation 107 

calculi 216 

oxidation of 115 

Urinary calculi 214 

sediments 175 

Urinometer 6 

Urobilin 88 

Uroerythrin 88 

Urohematin 89 

Urophain 89 

Vaginal mucin 46 

Variations in density 8 

Vegetable colors 14 and 99 

Vitellin 35 

Volhard's method 158 

Volume albumin test 32 

of urine 7 

Volumetric method for chlorides . 155 
phosphates 149 
sugar ... 67 
urea . . . .123 
uric acid . . 

108, in and 114 

Volumetric solutions 234 

Water, volume and density .... 225 

Waxy casts 190 and 192 

Weights, atomic 224 

and measures 223 

White corpuscles 182 

Wormseed 100 

Xanthir: 171 

bodies 167 

calculi 217 

Yeast cells in urine 199 

Zinc chloride creatinin 174 



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